Lipids and compositions for intracellular delivery of biologically active compounds

ABSTRACT

The present invention provides novel amino-lipids, compositions comprising such amino-lipids and methods of producing them. In addition, lipid nanoparticles comprising the novel amino-lipids and a biologically active compound are provided, as well as methods of production and their use for intracellular drug delivery.

CROSS-REFERENCE TO RELATED APPLICATIONS

1. Field of the Invention

The present invention provides novel amino-lipids, compositionscomprising such amino-lipids and methods of producing them. In addition,lipid nanoparticles (LNPs) comprising the novel amino-lipids and abiologically active compound are provided, as well as methods ofproduction and their use for intracellular drug delivery.

2. Background of the Invention

Lipid nanoparticles (LNPs), liposomes or lipoplexes are effective drugdelivery systems for biologically active compounds such as therapeuticproteins, peptides or nucleic acid based therapeutics, which areotherwise cell impermeable. Liposomal formulations have also beendeveloped for small molecule drugs with the aim to enrich the drug incertain tissues.

Drugs based on nucleic acids interact with a messenger RNA or a gene andhave to be delivered to the proper cellular compartment in order to beeffective. In particular double stranded nucleic acids, for exampledouble stranded RNA molecules (dsRNA) such as siRNAs, suffer from theirphysico-chemical properties that render them impermeable to cells. Upondelivery into the proper compartment, siRNAs block gene expressionthrough a highly conserved regulatory mechanism known as RNAinterference (RNAi). Typically, siRNAs are large in size with amolecular weight ranging from 12-17 kDa, and are highly anionic due totheir phosphate backbone with up to 50 negative charges. In addition,the two complementary RNA strands result in a rigid helix. Thesefeatures contribute to the siRNA's poor “drug-like” properties (NatureReviews, Drug Discovery 2007, 6:443). When administered intravenously,the siRNA is rapidly excreted from the body with a typical half-life inthe range of only 10 min. Additionally, siRNAs are rapidly degraded bynucleases present in blood and other fluids or in tissues, and have beenshown to stimulate strong immune responses in vitro and in vivo(Oligonucleotides 2009, 19:89).

By introduction of appropriate chemical modifications stability towardsnucleases can be increased and at the same time immune stimulation canbe suppressed. Conjugation of lipophilic small molecules to the siRNAsimproves the pharmacokinetic characteristics of the double stranded RNAmolecule. It has been demonstrated that these small molecule siRNAconjugates are efficacious in a specific down regulation of a geneexpressed in hepatocytes of rodents. However, in order to elicit thedesired biologic effect a large dose was needed (Nature 2004, 432:173).

With the advent of lipid nanoparticle formulations the siRNA dosesnecessary to achieve target knockdown in vivo could be significantlyreduced (Nature 2006, 441:111). Typically, such lipid nanoparticle drugdelivery systems are multi-component formulations comprising cationiclipids, helper lipids, lipids containing polyethylene glycol andcholesterol. The positively charged cationic lipids bind to the anionicnucleic acid, while the other components support a stable self-assemblyof the lipid nanoparticles.

To improve delivery efficacy of these lipid nanoparticle formulations,many efforts are directed to develop more appropriate cationic lipids.These efforts include high throughput generation of cationic lipidlibraries based on solvent- and protecting group free chemical reactionsuch as Michael additions of amines to acrylamides or acrylates (NatureBiotechnology 2008, 26:561) or ring-opening reactions with amines andterminal epoxides (PNAS 2010, 107:1854). Another strategy involvesstructure activity studies, e.g. systematic variation of the degree ofsaturation in the hydrophobic part (Journal of Controlled Release 2005,107:276) or the polar head group of the cationic lipid (NatureBiotechnology 2010, 28:172), resulting in an improved efficacy of theso-called stable nucleic acid-lipid particles (SNALP) technology(Current Opinion in Molecular Therapeutics 1999, 1:252).

Despites these efforts, improvements in terms of increased efficacy anddecreased toxicity are still needed, especially for lipid nanoparticlebased drug delivery systems intended for therapeutic uses. LNPsnaturally accumulate in the liver after intravenous injection into ananimal (Hepatology 1998, 28:1402). It has been demonstrated that genesilencing can be achieved in vivo in hepatocytes which account for themajority of the cells in the liver. Even the simultaneousdown-modulation of several target genes expressed in hepatocytes couldbe successfully achieved (PNAS 2010, 107:1854). However, evidence ofsuccessful gene regulation in other liver cell types is lacking.

SUMMARY OF THE INVENTION

The present invention provides novel amino-lipids, compositionscomprising the inventive amino-lipids, as well as methods of producingthem. In particular, compositions comprising the amino-lipids of theinvention that form lipid nanoparticles (LNPs) are provided, as well asmethods of producing and their use for the intracellular delivery ofbiologically active compounds, for example nucleic acids.

The methods of producing the amino-lipids provided herein areadvantageous compared to those known in prior art as the amino-lipidscan be produced with a higher yield and increased purity.

The lipid nanoparticles (LNPs) comprising the inventive amino-lipidssignificantly enhance the intracellular delivery of nucleic acids intohepatocytes compared to LNPs comprising lipids known in prior art. Inaddition, the lipid nanoparticles (LNPs) comprising the inventiveamino-lipids enable inhibition of gene expression in additional livercell types apart from hepatocytes, such as Kupffer cells, Stellate cellsand endothelial cells. Moreover, the lipid nanoparticles (LNPs)comprising the inventive amino-lipids are suitable for cell-typespecific delivery of nucleic acids into various organs in vivo,including jujunum, liver, kidney, lung and spleen. Importantly, theselipid nanoparticles can also be administered via the air ways enablinggene silencing in the lung.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Structures representing amino-lipids of the invention.

FIG. 2. Bar graph illustrating improved LNP composition of the inventionto reduce FVII mRNA levels in mice.

FIG. 3. Graph illustrating GFP mRNA levels after orotrachealinstillation of LNPs of the present invention into GFP transgenic mice(n=4). Individual siRNAs directed against the targets indicated belowthe bars were formulated into LNPs consisting of 50% KL25, 10% DSPC,38.5% cholesterol and 1.5% PEG2000-c-DOMG. 48 h post administrationanimals were killed, bronchoalveolar lavage fluid (BALD prepared and theGFP mRNA level determined using bDNA assay. Hatched bars represent GFPmRNA levels of animals treated with the unspecific siRNAs.

FIG. 4. Graph illustrating CD45 mRNA levels after orotrachealinstillation of LNPs of the present invention into GFP transgenic mice(n=4). Individual siRNAs directed against the targets indicated belowthe bars were formulated into LNPs consisting of 50% KL25, 10% DSPC,38.5% cholesterol and 1.5% PEG2000-c-DOMG. 48 h post administrationanimals were killed, bronchoalveolar lavage fluid (BALD prepared and theCD45 mRNA level determined using bDNA assay. Hatched bars represent CD45mRNA levels of animals treated with the unspecific siRNAs.

FIG. 5. Graph illustrating CD68 mRNA levels after orotrachealinstillation of LNPs of the present invention into GFP transgenic mice(n=4). Individual siRNAs directed against the targets indicated belowthe bars were formulated into LNPs consisting of 50% KL25, 10% DSPC,38.5% cholesterol and 1.5% PEG2000-c-DOMG. 48 h post administrationanimals were killed, bronchoalveolar lavage fluid (BALE) prepared andthe CD68 mRNA level determined using bDNA assay. Hatched bars representCD68 mRNA levels of animals treated with the unspecific siRNAs.

FIG. 6. Graph illustrating Clec7a mRNA levels after orotrachealinstillation of LNPs of the present invention into GFP transgenic mice(n=4). Individual siRNAs directed against the targets indicated belowthe bars were formulated into LNPs consisting of 50% KL25, 10% DSPC,38.5% cholesterol and 1.5% PEG2000-c-DOMG. 48 h post administrationanimals were killed, bronchoalveolar lavage fluid (BALf) prepared andthe Clec7a mRNA level determined using bDNA assay. Hatched barsrepresent Clec7a mRNA levels of animals treated with the unspecificsiRNAs.

FIG. 7. Graph illustrating Rein mRNA levels after siRNA treatmentemploying different LNPs of the present invention. The LNPs contained apool of five different siRNAs directed against five different targets(FVII, Rein, Clec4f, Tek, GFP). The siRNA dose was 0.5 mg/kg per siRNA(total siRNA dose 2.5 mg/kg). LNPs were dosed i.v. and 48 h post dosingmRNA levels were measured using bDNA assay. For comparison and shown hashashed bar, a benchmark LNP in which XTC2 substituted the amino-lipidwas included.

FIG. 8. Graph illustrating Clec4f mRNA levels after siRNA treatmentemploying different LNPs of the present invention. The LNPs contained apool of five different siRNAs directed against five different targets(FVII, Rein, Clec4f, Tek, GFP). The siRNA dose was 0.5 mg/kg per siRNA(total siRNA dose 2.5 mg/kg). LNPs were dosed i.v. and 48 h post dosingmRNA levels were measured using bDNA assay. For comparison and shown hashashed bar, a benchmark LNP in which XTC2 substituted the amino-lipidwas included.

FIG. 9. Graph illustrating Tek mRNA levels in liver after siRNAtreatment employing different LNPs of the present invention. The LNPscontained a pool of five different siRNAs directed against fivedifferent targets (FVII, Rein, Clec4f, Tek, GFP). The siRNA dose was 0.5mg/kg per siRNA (total siRNA dose 2.5 mg/kg). LNPs were dosed i.v. and48 h post dosing mRNA levels were measured using bDNA assay. Forcomparison and shown has hashed bar, a benchmark LNP in which XTC2substituted the amino-lipid was included.

FIG. 10. Graph illustrating Tek mRNA levels in liver after siRNAtreatment employing different LNPs of the present invention. The LNPscontained a pool of five different siRNAs directed against fivedifferent targets (FVII, Rein, Clec4f, Tek, GFP). The siRNA dose was 0.5mg/kg per siRNA (total siRNA dose 2.5 mg/kg). LNPs were dosed i.v. 5 and48 h post dosing mRNA levels were measured using bDNA assay. Forcomparison and shown has hashed bar, a benchmark LNP in which XTC2substituted the amino-lipid was included.

FIG. 11. Graph illustrating Tek mRNA levels in lung after siRNAtreatment employing different LNPs of the present invention. The LNPscontained a pool of five different siRNAs directed against fivedifferent targets (FVII, Rein, Clec4f, Tek, GFP). The siRNA dose was 0.5mg/kg per siRNA (total siRNA dose 2.5 mg/kg). LNPs were dosed i.v. and48 h post dosing mRNA levels were measured using bDNA assay. Forcomparison and shown has hashed bar, a benchmark LNP in which XTC2substituted the amino-lipid was included.

FIG. 12. Graph illustrating Tek mRNA levels in jejunum after siRNAtreatment employing different LNPs of the present invention. The LNPscontained a pool of five different siRNAs directed against fivedifferent targets (FVII, Reln, Clec4f, Tek, GFP). The siRNA dose was 0.5mg/kg per siRNA (total siRNA dose 2.5 mg/kg). LNPs were dosed i.v. and48 h post dosing mRNA levels were measured using bDNA assay. Forcomparison and shown has hashed bar, a benchmark LNP in which XTC2substituted the amino-lipid was included.

FIG. 13. Graph illustrating Reln mRNA expression levels of 2 individualanimals per LNP relative to saline treated animals 48 h post iv dosing.LNPs contained a pool of five different siRNAs directed against FVII,GFP, Rein, Clec4f and Tek each at a dose of 0.5 mg/kg (total siRNA dose2.5 mg/kg). LNPs were composed of 50 mol % amino-lipid designated by theKL numbers in the graphs below, 10 mol % DSPC, 38.5 mol % cholesteroland 1.5 mol % PEG-c-OMOG. For comparison and shown as hashed bar, abenchmark LNP in which XTC2 substituted the amino-lipid was included.

FIG. 14. Graph illustrating Clec4f mRNA expression levels of 2individual animals per LNP relative to saline treated animals 48 h postiv dosing. LNPs contained a pool of five different siRNAs directedagainst FVII, GFP, Rein, Clec4f and Tek each at a dose of 0.5 mg/kg(total siRNA dose 2.5 mg/kg). LNPs were composed of 50 mol % amino-lipiddesignated by the KL numbers in the graphs below, 10 mol % DSPC, 38.5mol % cholesterol and 1.5 mol % PEG-c-OMOG. For comparison and shown ashashed bar, a benchmark LNP in which XTC2 substituted the amino-lipidwas included.

FIG. 15. Graph illustrating Tek mRNA expression levels of 2 individualanimals per LNP relative to saline treated animals 48 h post iv dosing.LNPs contained a pool of five different siRNAs directed against FVII,GFP, Rein, Clec4f and Tek each at a dose of 0.5 mg/kg (total siRNA dose2.5 mg/kg). LNPs were composed of 50 mol % amino-lipid designated by theKL numbers in the graphs below, 10 mol % DSPC, 38.5 mol % cholesteroland 1.5 mol % PEG-c-OMOG. For comparison and shown as hashed bar, abenchmark LNP in which XTC2 substituted the amino-lipid was included.

FIG. 16. Graph illustrating Tek mRNA expression levels of 2 individualanimals per LNP relative to saline treated animals 48 h post iv dosing.LNPs contained a pool of five different siRNAs directed against FVII,GFP, Rein, Clec4f and Tek each at a dose of 0.5 mg/kg (total siRNA dose2.5 mg/kg). LNPs were composed of 50 mol % amino-lipid designated by theKL numbers in the graphs below, 10 mol % DSPC, 38.5 mol % cholesteroland 1.5 mol % PEG-c-OMOG. For comparison and shown as hashed bar, abenchmark LNP in which XTC2 substituted the amino-lipid was included.

FIG. 17. Graph illustrating Tek mRNA expression levels of 2 individualanimals per LNP relative to saline treated animals 48 h post iv dosing.LNPs contained a pool of five different siRNAs directed against FVII,GFP, Rein, Clec4f and Tek each at a dose of 0.5 mg/kg (total siRNA dose2.5 mg/kg). LNPs were composed of 50 mol % amino-lipid designated by theKL numbers in the graphs below, 10 mol % DSPC, 38.5 mol % cholesteroland 1.5 mol % PEG-c-OMOG. For comparison and shown as hashed bar, abenchmark LNP in which XTC2 substituted the amino-lipid was included.

DETAILED DESCRIPTION OF THE INVENTION

A. Amino-Lipids and Methods of Producing them.

The amino-lipids provided herein are produced by reductive amination ofa (poly)amine and an aliphatic carbonyl compound according to thegeneral reaction scheme:R′—NH₂+R—CHO→R′—N(CHR)₂R′—NH₂+R—CO—R→R′—N(CR₂)₂

The amino-lipids may be prepared by reacting the aliphatic carbonylcompound and the (poly)amine in the presence of a reducing agent.

In certain embodiments the aliphatic carbonyl compound is a ketone. Incertain embodiments the aliphatic carbonyl compound is an aldehyde.Typically, the (poly)amine has two to five nitrogen atoms in itsstructure. In certain embodiments the (poly)amine contains primaryand/or secondary and/or tertiary nitrogen atoms. Depending on thestructure of the (poly)amine, and the aliphatic carbonyl compoundemployed regioselective alkylations can be achieved. Particularly, whenketones are reacted with polyamines displaying primary and/or secondaryand/or tertiary nitrogens under reductive amination conditions selectivealkylations of primary nitrogens can be achieved. The present inventioncovers procedures of making amino-lipids of the following structures:

-   RNH(CH₂)_(x)NH₂,-   R₂N(CH₂)_(x)NH₂,-   R₂N(CH₂)_(x)NHR,-   R₂N(CH₂)_(x)NR₂,-   RNH[(CH₂)_(x)C_(w)H_(2w)NH)y(CH₂)_(z)]NH₂,-   RNH[(CH₂)_(x)C_(w)H_(2w)NR)y(CH₂)_(z)]NH₂,-   RNH[(CH₂)_(x)C_(w)H_(2w)NR)_(y)(C_(v)H_(2v)NH)_(u)(CH₂)_(z)]NH₂,-   R₂N[(CH₂)_(x)C_(w)H_(2w)NH)_(y)(CH₂)_(z)]NH₂,-   R₂N[(CH₂)_(x)C_(w)H_(2w)NR)_(y)(CH₂)_(z)]NH₂,-   R₂N[(CH₂)_(x)C_(w)H_(2w)NR)_(y)(C_(v)H_(2v)NH)_(u)(CH₂)_(z)]NH₂,-   RNH[(CH₂)_(x)(C₂H_(2w)NH)_(y)(CH₂)_(z)]NHR,-   RNH[(CH₂)_(x)(C₂H_(2w)NR)_(y)(CH₂)_(z)]NHR,-   RNH[(CH₂)_(x)(C_(w)H_(2w)NR)_(y)(C_(v)H_(2v)NH)_(u)(CH₂)_(z)]NHR,-   R₂N[(CH₂)_(x)(C_(w)H_(2w)NR)_(y)(CH₂)_(Z)]NHR,-   R₂N[(CH₂)_(x)(C_(w)H_(2w)NR)(CH₂)_(z)]NHR,-   R₂N[(CH₂)_(x)(C_(w)H_(2w)NH)_(y)(C_(v)H_(2v)NH)_(u)(CH₂)_(Z)]NR₂,-   N{[(CH₂)_(x)(C_(w)H_(2w)NH)_(y)(CH₂)_(z)]NHR}₃,-   N{[(CH₂)_(x)(C_(w)H_(2w)NH)_(y)(CH₂)_(z)]NR₂}₃,-   HN{[(CH₂)_(x)(C_(w)H_(2w)NH)_(y)(CH₂)_(z)]NHR}₂, and-   HN{[(CH₂)_(x)(C_(w)H_(2w)NH)_(y)(CH₂)_(z)]NR₂}₂,    wherein R is selected from alkyl, alkenyl or alkynyl carbon chains    ranging from C6 to C20. In certain embodiments these chains comprise    at least one, at least two or at least three sites of unsaturation,    for example one or more double bonds or triple bonds. In one    embodiment, R comprises at least one aromatic cycle, including for    example a heterocycle. In yet another embodiment, R may comprise at    least one heteroatom in the carbon chain, for example O, NH, NR′, S,    SS, wherein R′ is an acyl, alkyl, alkenyl or alkynyl group    consisting of two to 20 carbon atoms. In still another embodiment,    at least one hydrogen in the hydrocarbon chain R may be replaced by    F, Cl, Br, I. In one embodiment, w and v are independently 2, 3    or 4. In one embodiment, y and u are independently 0, 1, 2, 3 or 4.    In one embodiment, x and z are independently 2, 3 or 4.

In one aspect, the present invention provides cyclic amino-lipids of theformula (I):

whereinR¹ is independently selected from

-   -   —(CH₂)₂—N(R)₂,    -   —(CH₂)₂—N(R)—(CH₂)₂—N(R)₂, wherein R is independently selected        from —H, C6-40 alkyl, C6-40 alkenyl and C6-40 alkynyl, provided        that —N(R)₂ is not NH₂, and    -   C6-40 alkyl, and C6-40 alkenyl;        R² is C6-40 alkyl, C6-40 alkenyl, or C6-40 alkynyl;        m is 0 or 1; and        pharmaceutically acceptable salts thereof.

The term “C6-40 alkyl” as used herein means a linear or branched,saturated hydrocarbon consisting of 6 to 40 carbon atoms, preferably of6 to 30 carbon atoms, most preferably of 6 to 20 carbon atoms.Especially preferred are alkyl groups containing 10, 14 or 15 carbonatoms.

The term “C6-40 alkenyl” as used herein means a linear or branched,unsaturated hydrocarbon consisting of 6 to 40 carbon atoms, preferablyof 6 to 30 carbon atoms, most preferably of 6 to 15 carbon atoms. In oneembodiment the C6-40 alkenyl groups comprise 1 to 4 double bonds,preferably between 1 to 3 double bonds, most preferably 1 or 2 doublebonds.

The term “C6-40 alkynyl” as used herein means a linear or branched,unsaturated hydrocarbon consisting of 6 to 40 carbon atoms, preferablyof 6 to 30 carbon atoms, most preferably of 6 to 20 carbon atoms. In oneembodiment the C₆-40 alkynyl groups comprise 1 to 4 triple bonds,preferably 1 to 3 triple bonds, most preferably 1 or 2 triple bonds.

In another embodiment there are provided the cyclic amino-lipidsselected from

Preferred therein are the cyclic amino-lipids selected from

In one aspect, the present invention provides linear amino-lipids of theformula (II):

wherein

-   -   R³ is independently selected from C1-40 alkyl or C6-40 alkenyl,        wherein up to 4 carbon atoms may be replaced by a heteroatom        selected from oxygen or nitrogen;    -   R⁴ is selected from C12-40 alkyl or C6-40 alkenyl, wherein up to        4 carbon atoms may be replaced by a heteroatom selected from        oxygen or nitrogen;    -   R⁵ is selected from hydrogen, C12-30 alkyl and C6-40 alkenyl;    -   R⁶ is selected from hydrogen and C₁₋₁₂ alkyl;    -   n is 1, 2, or 3; and    -   k is 1, 2, 3 or 4.

In a preferred embodiment, a linear amino-lipids of the formula (II) isprovided wherein

-   -   R³ is independently selected from C1-, C12-, C14-, C27-, C30-        and C37-alkyl, wherein 1 or 2 carbon atoms can be optionally        replaced by an oxygen or a nitrogen atom;    -   R⁴ is selected from C12-, C14-, C27-, C30- and C37-alkyl,        wherein 1 or 2 carbon atoms can be optionally replaced by an        oxygen or a nitrogen atom;    -   R⁵ is selected from hydrogen, C12-, C14- and C30-alkyl, in which        one carbon atom can be optionally replaced by a nitrogen atom;    -   R⁶ is selected from hydrogen, C1- and C12-alkyl; and    -   n and k have the meanings given in claim 1.

In one embodiment there are provided the linear amino-lipids selectedfrom

Preferred therein is the linear amino-lipid

In yet another embodiment an amino-lipid of formula

is provided.

In yet another embodiment an amino-lipid of formula

is provided.

In yet another embodiment an amino-lipid of formula

is provided.

In particular embodiments the amino-lipids of the present inventioncomprise Nitrogen atoms that are protonated depending on the pH of theenvironment, preferably at least one Nitrogen atom is positively chargedat physiological pH or below. The extent of pH dependent protonation iseffected by an equilibrium reaction and hence not the entire, but onlythe predominant lipid species is positively charged. At physiological pHat least one of the nitrogen atoms in the lipid structure is protonated.

As used herein, the term “(poly)amine” refers to a saturated hydrocarbonlinear or branched wherein 2 to 5 Carbon atoms are replaced by Nitrogen.Preferably said (poly)amine comprises two to five nitrogen atoms.Preferred therein are (poly)amines that comprise amine Nitrogens thatare separated by 2 and/or 3 and/or 4 carbon atoms. Non-limiting examplesof suitable (poly)amines are ethylenediamine, diethylenetriamine,triethylenetetramine, tetraethylenepentamine, tris-(2-aminoethyl)-amine,3-dimethylamino-1-propylamine, spermine, spermidine,2,2′-(ethylenedioxy)-bis(ethylamine). The term “aliphatic carbonylcompound” as used herein refers to a compound R—CO—R′, wherein R is aketone or an aldehyde comprising of alkyl and/or alkenyl and/or alkynylgroups and R′ is H or a ketone or an aldehyde comprising of alkyl and/oralkenyl and/or alkynyl groups.

The term “reducing agent” as used herein refers to a reagent thatenables the reduction of the iminium ion intermediate in reductiveamination reactions. Examples of such reagents include, but are notlimited to hydride reducing reagents such as sodium cyanoborohydride,sodium triacetoxyborohydride and sodium borotetrahydride. Catalytichydrogenation with metal catalyst such as nickel, palladium or platinumcan also be used for this purpose. As used herein, the term “lipid”refers to amphiphilic molecules comprising a polar, water soluble“headgroup” and a hydrophobic “tail”. The headgroup preferably consistsof a pH dependent charged group such as an amine. The tail preferablycomprises aliphatic residues. Lipids can be of natural origin or ofsynthetic origin. Examples include, but are not limited to, fatty acids(e.g. oleic acid, lineolic acid, stearic acid), glycerolipids (e.g.mono-, di-, triglycerols such as triglycerides), phospholipids (e.g.phosphatidylethanolamine, phosphatidylcholine), and sphingolipids (e.g.sphingomyelin)

As used herein, the term “amino-lipid” refers to lipids having at leastone of the Nitrogen atoms incorporated in at least one fatty acid chain.This fatty acid chain may be an alkyl, alkenyl or alkynyl carbon chain.Lipids containing carbon chain lengths in the range from C10 to C20 arepreferred. It is understood that the fatty acid portion of theamino-lipid of the present invention is incorporated through the use ofsuitable carbonyl compounds such as aldehydes (R—CHO) and ketones(R—CO—R). Through the use of asymmetrical ketones (R—CO—R′)corresponding unsymmetrical substituted lipids can be prepared.Likewise, through the use of carbonyl ethers, esters, carbamates andamides and suitable reducing agents the corresponding amino-lipids areaccessible.

The term “cyclic amino-lipid” as used herein refers to an amino-lipid ofthe general formula (I).

The term “linear amino-lipid” as used herein refers to an amino-lipid ofthe general formula (II).

In another aspect, novel amino-lipids can be prepared by reacting asuitable (poly)amine with a carbonyl compound in the presence of areducing agent to form a cyclic or linear amino-lipid.

In certain embodiments the alkyl, alkenyl and alkynyl groups as coveredin the present invention contain 2 to 20 carbon atoms. In certain otherembodiments these groups consists of 2 to 10 carbon atoms. In yet otherembodiments the alkyl, alkenyl and alkynyl groups employed in thisinvention contain 2 to 8 carbon atoms. In still other embodiments thesegroups contain two to six carbon atoms. In yet other embodiments thealkyl, alkenyl and alkynyl groups of the invention contain 2 to fourcarbon atoms.

The term “alkyl” as used herein means a chain of saturated hydrocarbonsthat is aliphatic, branched or cyclo-aliphatic. Saturated aliphatichydrocarbons include methyl, ethyl, n-propyl, n-butyl and the like.Saturated branched alkyls include isopropyl, isobutyl, tert-butyl andthe like. Representative cyclo-aliphatic alkyls include cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl and the like.

The term “alkenyl” denotes a chain of hydrocarbons that has at least onecarbon-carbon double bond. For example alkenyl groups include ethenyl,propenyl, butenyl, isopropylidene and the like. The term also coverscyclic alkenyls such as cyclopropenyl, cyclobutenyl, cyclopentenyl andthe like.

The term “alkynyl” denotes a chain of hydrocarbons that has at least onecarbon-carbon triple bond. Exemplary alkynyl groups include ethynyl,propynyl, butynyl and the like. The term also covers cyclic alkynylssuch as cyclopentynyl, cyclohexynyl, and the like.

The term “acyl” refers to any alkyl, alkenyl or alkynyl group that islinked through a carbonyl group. For example, acyl groups are—(CO)-alkyl, —(CO)-alkenyl and —(CO)-alkynyl.

B. Lipid Nanoparticles (LNPs) Comprising the Inventive Amino-Lipids

Also provided herein are compositions comprising the amino-lipids of theinvention that form lipid nanoparticles (LNPs). As used herein, the term“lipid nanoparticles” includes liposomes irrespective of theirlamellarity, shape or structure and lipoplexes as described for theintroduction of pDNA into cells (PNAS, 1987, 84, 7413). These lipidnanoparticles can be complexed with biologically active compounds suchas nucleic acids and are useful as in vivo delivery vehicles. Preferablysaid in vivo delivery is cell-type specific.

In one embodiment, said lipid nanoparticles comprise one or moreamino-lipids of the invention described above and may furthermorecomprise additional lipids and other hydrophobic compounds such assterol derivatives, e.g. cholesterol. Those additional components of alipid nanoparticle of the present invention serve various purposes suchas aiding manufacturing and storage stability as well as modulation ofthe biodistribution. Biodistribution may also be modulated byincorporation of targeting ligands conjugated to the lipids part of thelipid nanoparticle. Specific examples of additional components of thelipid nanoparticles are given below.

In one embodiment, lipid nanoparticles are provided that comprise theamino-lipids described above and one or more additional lipids.Additional lipids suitable to be incorporated into the lipidnanoparticles of the invention comprise cationic lipids, helper lipidsand PEG lipids. Hence in one embodiment lipid nanoparticles are providedthat comprise the amino-lipids described above and one or moreadditional lipids selected from the group of cationic lipid, helperlipid and PEG lipid. “Cationic lipids” as used herein refers to anylipid comprising a quaternary amine and are consequently permanentlypositively charged. The term “quaternary amine” as used herein refers toa nitrogen atom having four organic substituents. For example, thenitrogen atom in Tetramethylammonium chloride is a quaternary amine.

Examples of cationic lipids comprising a quaternary amine include, butare not limited to, N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammoniumchloride (“DOTAP”), N,N,-Distearyl-N,N-dimethylammonium bromide(“DDBA”), 1-methyl-4-(cis-9-dioleyl)-methylpyridinium-chloride(“SAINT-solid”), N-(2,3-dioleyloxy)propyl)-N,N,N-triethylammoniumchloride (“DOTMA”), N,N-dioleyl-N,N-dimethylammonium chloride (“DODAC”),(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (“DIMRIE”) and the like.

“Helper lipids”, as used herein are preferably neutral zwitterioniclipids. Examples of preferred helper lipids used in this invention are1,2-distearoyl-sn-glycero-3-phosphocholine (“DSPC”),1,2-dipalmitoyl-sn-glycero-3-phosphocholine (“DPPC”), or any relatedphosphatidylcholine such as natural sphingomyelin (“SM”) and syntheticderivatives thereof such as1-oleoyl-2-cholesteryl-hemisuccinoyl-sn-glycero-3-phosphocholine(“OChemsPC”). Other preferred helper lipids include1,2-dileoyl-sn-3-phosphoethanolamine (“DOPE”),1,2-diphytanoyl-sn-glycero-3-phosphoethanol-amine (“ME 16:0 PE”).

In one embodiment, LNPs contain uncharged lipids modified withhydrophilic polymers, e.g. polyethylene glycol (herein also referred toas “PEG-lipids”) to stabilize the lipid nanoparticle and to avoidaggregation. The polyethylene glycol (PEG) size can vary fromapproximately 1 to 5 approximately kDa. Depending on the relativeamounts of these molecules in the formulation and the length of thehydrocarbon chain, the PEG-lipid can influence the pharmacokineticcharacteristics, biodistribution, and efficacy of a formulation. PEGlipids having relatively short lipid hydrocarbon chains of about 14carbons dissociate from the LNP in vivo in plasma with a half-life ofless than 1 h. In contrast, a PEG lipid with a relatively long lipidhydrocarbon chain length of about 18 carbons circulates fully associatedwith the formulation for several days. Hence, in one preferredembodiment, said PEG lipid comprises a lipid hydrocarbon chain of 12 to20 carbon atoms, 14 to 18 carbon atoms, preferably of 14 carbon atoms.

Typically, the concentration of the PEG-lipid is about 0.5 to 10 mol %.Examples of suitable PEG modified lipids include pegylated ceramideconjugates, pegylated distearoylphosphatidyl-ethanolamine (PEG-DSPE).Other compounds that can be used to stabilize lipid nanoparticlesinclude gangliosides (GM_(t), GM3, etc.). Preferred PEG lipids have aPEG size ranging from about 1 to about 2 KDa. Specific examples aremethoxy-polyethyleneglycol-carbamoyl-dimyristyloxy-propylamine(PEG2000-c-DMA), and(α-(3′-(1,2-dimyristoyl-3-propanoxy)-carboxamide-propyl]-ω-methoxy-polyoxyethylene(PEG2000-c-DOMG).

In one embodiment lipid nanoparticles are provided that comprise theamino-lipids described above and one or more hydrophobic small molecule.The term “hydrophobic small molecule” as used herein refers to acompound with a molecular weight of about 300 to about 700 Da comprising2 or more carbon- or heterocycles providing a rigid core structure.Preferably said hydrophobic small molecule is selected from the group ofsterols such as cholesterol or stigmasterol or a hydrophobic vitaminsuch as tocopherol. In a preferred embodiment said hydrophobic smallmolecule is cholesterol.

In one embodiment the lipid nanoparticle comprises an amino-lipid of thepresent invention, one or more additional lipids selected from the groupof cationic lipid, helper lipid and PEG lipid, and a hydrophobic smallmolecule selected from the group of a sterol or a hydrophobic vitamin.In one embodiment said lipid nanoparticle comprises an amino-lipid ofthe present invention, a helper lipid selected from DSPC or DPPC,PEG-DOMG and a hydrophobic small molecule selected from the group of asterol or a hydrophobic vitamin.

In one embodiment the lipid nanoparticle comprises an amino-lipid of thepresent invention, a helper lipid, a PEG modified lipid and cholesterol.In preferred embodiments the molar ratios of these components are 30-70%amino-lipid, 0-60% helper lipid, 0.1-10% PEG lipid and 0-50%cholesterol. More preferred lipid nanoparticle compositions comprise theabove mentioned components in a molar ratio of about 40% to 60%amino-lipid, 0 to 20% helper lipid, 0.1% to 5% PEG lipid and 30 to 50%cholesterol. In certain other embodiments lipid nanoparticles areprovided that do not comprise cholesterol. These formulations contain upto about 60 mol % of at least one helper lipid. Preferred helper lipidsin these lipid nanoparticles are DSPC, SM, DOPE, 4ME16:0 PE.

In one embodiment the lipid nanoparticle comprises a cyclic amino-lipidof the present invention, DSPC or SM, PEG-c-DOMG and cholesterol.Preferably, said cyclic amino-lipid has the structure of

Preferred molar ratios of these components are about 50% of said cyclicamino-lipid, about 10% helper lipid, about 38% cholesterol and about 2%of the PEG lipid. Preferred N/P ratios range from approximately 6.9 toapproximately 8.4.

In another embodiment cholesterol free lipid nanoparticles are provided.These comprise a cyclic amino-lipid, the helper lipids DSPC and DOPE, aswell as the PEG-lipid PEG-c-DMOG. LNPs comprising these components arenot taken up by Kupffer cells in the liver, but mediate functional drugdelivery to hepatocytes, stellate cells and endothelial cells.Preferably said cyclic amino-lipid has the structure of

In yet another embodiment cholesterol free lipid nanoparticles comprisejust one helper lipid. In this case a preferred lipid nanoparticlecontains a cyclic amino-lipid, the helper lipid 4ME 16:0 PE andPEG-c-DMOG. LNPs comprising these components are barely taken up byhepatocytes, but mediate functional drug delivery to Kupffer cells,stellate cells and endothelial cells.

Preferably said cyclic amino-lipid has the structure of

In one embodiment the lipid nanoparticle comprises an amino-lipid of thepresent invention, DSPC, a PEG lipid such as PEG-c-DOMG and cholesterol.Preferably, said amino-lipid has the structure of

Preferred molar ratios of these components are 40% to 60% of saidamino-lipid, about 0% to 20% helper lipid, about 38% cholesterol andapproximately 2% of a PEG 2000 lipid. The N/P ratio preferably is atleast about 15:1, more preferably at least about 10:1, even morepreferably at least about 7:1, and most preferably at least about 5:1.LNPs comprising these compositions are particularly well suited tofunctional deliver nucleic acids into endothelial cells of variousorgans.

In another embodiment the lipid nanoparticle comprises an amino-lipid ofthe present invention, DSPC, a PEG lipid such as PEG-c-DOMG andcholesterol. Said amino-lipid has the structure of

Preferred molar ratios of these components are 40% to 60% of saidamino-lipid, about 0% to 20% helper lipid, about 30% to 40% cholesteroland about 0.5% to about 2% of a PEG 2000 lipid. The N/P ratio preferablyis at least about 8:1, more preferably at least about 15:1 and mostpreferably at least about 10:1.

In another preferred embodiment the lipid nanoparticle comprises anamino-lipid of the present invention, DSPC, a PEG lipid such asPEG-c-DOMG and cholesterol. Said amino-lipid has the structure of

In another preferred embodiment the lipid nanoparticle comprises acyclic amino-lipid of the present invention, DSPC, a PEG lipid such asPEG-c-DOMG and cholesterol. Said amino-lipid has the structure of

In another preferred embodiment the lipid nanoparticle comprises acyclic amino-lipid of the present invention, DSPC, a PEG lipid such asPEG-c-DOMG and cholesterol. Said amino-lipid has the structure of

In one embodiment, the lipid nanoparticles described above are complexedwith a biologically active compound. The term “complexed” as used hereinrelates to the non-covalent interaction of the biologically activecompound with specific components of the lipid nanoparticle. In case ofa nucleic acid as the biologically active compound the negativelycharged phosphate backbone of the nucleic acid interacts with thepositively charged amino-lipid. This interaction supports the stableentrapment of the nucleic acid into the LNP.

The term “biologically active compound” as used herein refers to aninorganic or organic molecule including a small molecule, peptide (e.g.cell penetrating peptides), carbohydrate (including monosaccharides,oligosaccharides, and polysaccharides), protein (includingnucleoprotein, mucoprotein, lipoprotein, synthetic polypeptide, or asmall molecule linked to a protein, glycoprotein), steroid, nucleicacid, lipid, hormone, or combination thereof, that causes a biologicaleffect when administered in vivo to an animal, including but not limitedto birds and mammals, including humans. Preferably said biologicallyactive compound is negatively charged.

In one embodiment the lipid nanoparticles described above are complexedwith a biologically active compound selected from the group of smallmolecule, peptide, protein, carbohydrate, nucleic acid, or lipid.Preferably said biologically effect is a therapeutic effect.

The term “nucleic acid” as used herein means an oligomer or polymercomposed of nucleotides, e.g., deoxyribonucleotides or ribonucleotides,or compounds produced synthetically (e.g., PNA as described in U.S. Pat.No. 5,948,902 and the references cited therein) which can hybridize withnaturally occurring nucleic acids in a sequence specific manneranalogous to that of two naturally occurring nucleic acids, e.g., canparticipate in Watson-Crick base pairing interactions. Non-naturallyoccurring nucleic acids are oligomers or polymers which containnucleobase sequences which do not occur in nature, or species whichcontain functional equivalents of naturally occurring nucleobases,sugars, or inter-sugar linkages, like peptide nucleic acids (PNA),threose nucleic acids (TNA), locked nucleic acids (LNA), or glycerolnucleic acids (GNA). This term includes oligomers that contain thenaturally occurring nucleic acid nucleobases adenine (A), guanine (G),thymine (T), cytosine (C) and uracil (U), as well as oligomers thatcontain base analogs or modified nucleobases. Nucleic acids can derivefrom a variety of natural sources such as viral, bacterial andeukaryotic DNAs and RNAs. Other nucleic acids can be derived fromsynthetic sources, and include any of the multiple oligonucleotides thatare being manufactured for use as research reagents, diagnostic agentsor potential and definite therapeutic agents. The term includesoligomers comprising a single strand nucleic acid or a double strandnucleic acid. Examples of nucleic acids useful therein are miRNA,antisense oligonucleotides, siRNA, immune-stimulatory oligonucleotides,aptamers, ribozymes, or plasmids encoding a specific gene or siRNA.

As used herein, the term “peptide” is used to refer to a natural orsynthetic molecule comprising two or more amino acids linked by an amidebond consisting of the carboxylic acid group of one amino acid and theamino group by the other amino acid. A peptide is not limited by thenumber of amino acids and thus it can include polypeptides and proteins.

Below embodiments are exemplified for the complexation and delivery ofnucleic acids. It is understood that these embodiments are alsoapplicable for any other biologically active compound.

The complex comprising lipid nanoparticles and one or more nucleic acidare characterized by the following parameters: (1) nucleic acid to totallipid ratio; (2) nucleic acid to amino-lipid ratio; (3) encapsulationefficacy; (4) particle size; (5) particle size distribution and (6) zetapotential.

The nucleic acid to total lipid ratio is the amount of the nucleic acidin a defined volume divided by the amount of total lipid in the samevolume. Total lipid refers to all components in the particleformulations except for the nucleic acid. The ratio may be expressed ona mole per mole or weight by weight basis.

The nucleic acid to amino-lipid ratio is the amount of the nucleic acidin a defined volume divided by the amount of amino-lipid in the samevolume. The ratio may be expressed on a mole per mole or weight byweight basis; but is usually expressed as nitrogen to phosphorus (N/P)ratio. The (N/P) ratio is characterized by the number of positivelycharged nitrogen atoms present in the amino-lipid divided by the numberof negatively charged phosphorus atoms present in the nucleic acid.Encapsulation efficacy is defined as the percentage of nucleic acid thatis encapsulated or otherwise associated with the lipid nanoparticle. Theencapsulation efficiency is usually determined by quantifying the amountof the nucleic acid in solution before and after breaking up the lipidnanoparticle by suitable organic solvents or detergents. A highencapsulation efficiency is a desirable feature of nucleic acid lipidnanoparticles particularly because of considerations regarding cost ofgoods.

The particle size of lipid nanoparticles is typically measured bydynamic light scattering. Sizes of lipid nanoparticles in a givenformulation are typically distributed over a certain range The size oflipid nanoparticles is typically expressed as the mean particle size orthe Z_(average) value. The particle size distribution of lipidnanoparticles is expressed as the polydispersity index (PI). Particlesin the size range of 30 to 300 nm are considered advantageous for invivo applications. Lipid nanoparticles with a mean particle size lessthan approximately 150 nm are advantageous, in particular to assesstissues characterized by a leaky vasculature as is the case for tumortissue or liver. Lipid nanoparticles with a mean particle size greaterthan approximately 150 nm are advantageous, in particular to assessmacrophages.

Nucleic acid lipid nanoparticles are also characterized by their surfacecharge as measured by the zeta (0-potential. The basis of themeasurement is the movement of particles in an electrical field asmeasured by dynamic light scattering. Particles with a near to neutralsurface charge are advantageous for in vivo applications and are thuspreferred herein.

Particularly because of cost of goods and manufacturing reasons a highencapsulation efficiency of the nucleic acids complexed with the lipidnanoparticles is desirable. Particles in the size range of 30 to 300 nmwith near to neutral surface charge are known to be advantageous for invivo applications.

C. Methods of Producing Lipid Nanoparticles (LNPs) Comprising theInventive Amino-Lipids and Complexes Thereof with Biologically ActiveCompounds.

In general, any method known in the art can be applied to prepare thelipid nanoparticles comprising one or more amino-lipids of the presentinvention and to prepare complexes of biologically active compounds andsaid lipid nanoparticles. Examples of such methods are widely disclosed,e.g. in Biochimica et Biophysica Acta 1979, 557:9; Biochimica etBiophysica Acta 1980, 601:559; Liposomes: A practical approach (OxfordUniversity Press, 1990); Pharmaceutica Acta Helvetiae 1995, 70:95;Current Science 1995, 68:715; Pakistan Journal of PharmaceuticalSciences 1996, 19:65; Methods in Enzymology 2009, 464:343.

Below embodiments are exemplified for the complexation and delivery ofnucleic acids. It is understood that these embodiments are alsoapplicable for any other biologically active compound.

In one embodiment, the components of the lipid nanoparticles as outlinedabove are mixed in a solvent that is miscible with water, such asmethanol, ethanol isopropanol or acetone. Preferred solvents arealcohols, most preferable ethanol. In most preferred embodiments thesolvent is commercially available ethanol. In certain embodiments thelipid mixture consists of the above components in a molar ratio of about30 to 70% amino-lipid: 0 to 60% helper lipid: 0.1 to 10% PEG-lipid and 0to 50% cholesterol. More preferred lipid nanoparticle compositionscomprise the above mentioned components in a molar ratio of about 40% to60% amino-lipid, 0 to 20% helper lipid, 0.1% to 5% PEG lipid and 30 to50% cholesterol. In certain other embodiments lipid nanoparticles lackcholesterol. These formulations contain up to about 60 mol % of at leastone helper lipid. Preferred helper lipids in these lipid nanoparticlesare DSPC, SM, DOPE, 4ME16:0 PE.

In one embodiment, the nucleic acid is dissolved in an aqueous buffer.The pH of the buffer is such that at least one of the nitrogen atoms ofthe amino-lipids of the present invention will become protonated uponmixing the aqueous nucleic acid solution with the solution comprisingthe components of the lipid nanoparticles. Examples of appropriatebuffers include, but are not limited to acetate, phosphate, citrate,EDTA and MES. Preferred concentration of the buffers are in the range ofabout 1 to about 500 mM. Typically the concentration of the nucleic acidin the aqueous buffer is in the range of about 0.1 to about 250 mg/mL,more preferably from about 0.5 to about 150 mg/mL.

The solution comprising the components of the lipid nanoparticles isthen combined with the buffered aqueous solution of the nucleic acid.Acidic pH is preferred, particularly a pH below 6.8, more preferably apH below 5.4 and most preferably about 4.0. Optionally, the entiremixture may be sized according to known methods, e.g. by extrusion.Particles with a mean diameter of preferably 40 to 170 nm, mostpreferably of about 50 to 120 nm are generated. Subsequently, the pH isneutralized yielding an at least partially surface-neutralized nucleicacid lipid nanoparticle complex. Due to the fact that amino-lipids ofthe present invention have at least two nitrogen atoms, pKa values candiffer substantially. Formation of complexes with nucleic acids is mostsupported at low pH in the range of about 3 to about 5. At a pH of about7, at least partial surface neutralization is achieved.

In one embodiment, the ratio of lipid:nucleic acid is at least about2:1, at least about 3:1, at least about 4:1, at least about 5:1, atleast about 6:1, at least about 7:1, at least about 8:1, at least about10:1, at least about 15:1.

Techniques to combine the solutions comprising the components of thelipid nanoparticles and the buffered aqueous solution of the nucleicacid can vary widely and may be dependent on the scale of production.Preparations in the range of a few mL may be made by pipetting onesolution into the other followed by mixing with e.g. a vortex mixture.Larger volumes can be preferentially prepared by a continuous mixingmethod using pumps, e.g. a piston pump such as Pump 33 (HarvardApparatus) or most preferably HPLC pumps such as AKTA pumps (GEHealthcare). With the aid of such pumps the two solutions are pumped outof their individual reservoirs and combined by delivering the fluidsthrough a suitable connector piece or mixing chamber. By varying theconcentrations of the two solutions and their flow rates, the mean sizeof the resulting lipid nanoparticles can be controlled within a certainrange. Preferably, the compositions provided herein are sized to a meandiameter from about 50 nm to about 200 nm, more preferably about 50 nmto about 150 nm and most preferably about 50 nm to 120 nm.

In certain embodiments, methods of the present invention furthercomprise a processing step that ensures the substantial removal of thesolvent that was used to dissolve the lipid mixture and to exchange thebuffer used to dissolve the therapeutically active agent. Suitabletechniques to carry out this processing step include, but are notlimited to diafiltration or tangential flow filtration. For bufferexchange a physiologically compatible buffer such as phosphate or HEPESbuffered saline with a pH of about 7.4 or 5% dextrose solution (DSW) isused.

Optionally, nucleic acid lipid nanoparticles can be produced using thelipid film hydration method followed by extrusion. In this case, thecomponents of the lipid nanoparticle, i.e. one of the amino-lipids asdescribed in the present invention, a helper lipid, a PEG-lipid (e.g.PEG-c-DOMG) and a sterol, e.g. cholesterol, are dissolved in an organicsolvent such as chloroform. The solvent is evaporated yielding a thinlipid film, which is subsequently hydrated with an aqueous buffercontaining the therapeutically active agent to form the desired lipidnanoparticle. Alternatively, the lipid film is hydrated with buffer andthe nucleic acid is added in a subsequent incubation step.

D. Pharmaceutical Compositions and Medical Uses.

In another object the present invention relates to a pharmaceuticalcomposition comprising the amino-lipids of the invention. Preferablysaid pharmaceutical composition comprises the lipid nanoparticles of thepresent invention and a biologically active compound. In one embodimentsaid biologically active compound is selected from the group of a smallmolecule, a peptide, a protein or a nucleic acid. In a preferredembodiment, said biologically active compound is a nucleic acid.Examples of nucleic acids useful therein are miRNA, antisenseoligonucleotides, siRNA, immune-stimulatory oligonucleotides, aptamers,ribozymes, or plasmids encoding a specific gene or siRNA.

The pharmaceutical compositions provided herein may additionally containadjuvants such as preservatives, wetting agents, emulsifying agents anddispersing agents. Prevention of presence of microorganisms may beensured both by sterilization procedures, supra, and by the inclusion ofvarious antibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol, sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

Regardless of the route of administration selected, the lipidnanoparticles of the present invention, which may be used in a suitablehydrated form, and/or the pharmaceutical compositions of the presentinvention, are formulated into pharmaceutically acceptable dosage formsby conventional methods known to those of skill in the art.

The phrases “administration” and “administered” as used herein meansmodes of administration other than enteral and topical administration,usually by injection, and includes, without limitation, intravenous,intramuscular, intraarterial, intrathecal, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal,epidural and intrasternal injection and infusion.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, the route of administration, the time ofadministration, the rate of excretion of the particular compound beingemployed, the duration of the treatment, other drugs, compounds and/ormaterials used in combination with the particular compositions employed,the age, sex, weight, condition, general health and prior medicalhistory of the patient being treated, and like factors well known in themedical arts.

The pharmaceutical composition must be sterile and fluid to the extentthat the composition is deliverable through syringe or infusiontechniques. In addition to water, the carrier is preferably an isotonicsugar solution and most preferably an isotonic buffered saline solution.

Proper fluidity can be maintained, for example, by use of coating suchas lecithin, by maintenance of required particle size and by use ofsurfactants. In many cases, it is preferable to include isotonic agents,for example, sugars, polyalcohols such as mannitol or sorbitol, andsodium chloride in the composition.

In another object the present invention relates to the use of the lipidnanoparticles of the invention complexed to a biologically activecompound as a medicament for treatment of a disease. In one embodimentthe biologically active compound is a nucleic acid that may comprise asingle strand or a double strand DNA or RNA which may or may not bechemically modified. Examples of nucleic acids useful therein are miRNA,short interfering RNA (siRNA), antisense oligonucleotides,immune-stimulatory oligonucleotides, aptamers, ribozymes, or plasmidsencoding a specific gene or a siRNA. In particular embodiments thebiologically active compound is a short interfering RNA (siRNA) and iscomplexed with the lipid nanoparticles of the present invention, thusenabling intracellular delivery of said biologically active compound.

In one embodiment the present invention provides a method of treating adisease that is caused by the over-expression of one or several proteinsin a subject, said method comprising administration of the apharmaceutical composition of the present invention to said subject. Thepharmaceutical composition comprises the LNP of the invention and abiologically active compound selected from the group of siRNA, miRNA,antisense oligonucleotides, ribozyme or a plasmid encoding for an siRNA,all being able to interfere with the expression of the disease causingprotein(s).

In another embodiment, the present invention provides a method oftreating a disease that is caused by a reduced expression or asuppressed expression of one or several proteins in a subject, saidmethod comprising administration the pharmaceutical composition of thepresent invention to the subject. The pharmaceutical compositioncomprises the LNP of the invention and a biologically active compoundselected from the group of a plasmid encoding for the correspondingprotein(s) or a nucleic acid that interferes with the suppressormolecule.

In yet another embodiment, the present invention provides for a methodof generating an immune response in a subject upon administration of apharmaceutical composition of the present invention to said subject. Thepharmaceutical composition comprises the LNP of the invention and abiologically active compound, wherein the biologically active compoundis an immune-stimulatory nucleic acid such as a CpG oligonucleotide. Asused herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, preventing occurrence or recurrence of disease, alleviation ofsymptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, antibodies ofthe invention are used to delay development of a disease or to slow theprogression of a disease.

EXAMPLES

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

Example 1 Amino-Lipids

Examples of amino-lipids of the present invention synthesized byreaction of an amine and a carbonyl compound under reductive aminationconditions are listed in Table 1. Solvents and reagents were purchasedfrom Sigma Aldrich (Taufkirchen, Germany) or TCI Europe (Eschborn,Germany) and were used as received (FIG. 1).

A) General Synthesis Procedure for Amino-Lipids Generated by Reaction ofAmines and Aldehydes.

The aldehydes needed for the preparation of KL5, KL6 KL7, KL8, KL12,KL15, KL16, KL34, KL35, KL37 were prepared by oxidation of thecorresponding alcohols using 2-Iodoxybenzoic acid (IBX) according to thefollowing general procedure:

The alcohol was dissolved in anhydrous ethyl acetate (EtOAc, 10 mL/1.0mmol). IBX (1.1 eq) was added to form a suspension. The mixture wasstirred briskly and refluxed at 80° C. under Argon. DMSO (2.2 eq.) wasadded via a syringe and the suspension was refluxed for 1.5 h. Theinsoluble o-iodobenzoic acid by-product was filtered off. The solventwas removed under reduced pressure and the crude product purified byflash-chromatography (Hexan-Hexcan/EtOAc=10:1, Rf=0.35 inHexan/EtOAc=5:1). The final products were characterized by HPLC and massspectrometry.

Ether containing alcohols were prepared following a published procedure(Bioconjugate Chemistry 2006, 19:1283). The subsequent oxidation to thecorresponding aldehyde was again accomplished with IBX according to theprocedure given above.

The aldehydes needed for the preparation of KL52 and KL56 weresynthesized by oxidation of the corresponding alcohols employingpyridinium chlorochromate (PCC) in Dichloromethane according to standardprocedures (Synthesis, 1982, 245). Other aldehydes are commerciallyavailable. The amine in KL12, KL22, KL33, KL34 and KL35 was madepursuing a published synthesis route (PNAS 2010, 5:1864).

B) Preparation of N-peralkylated Amino-Lipids.

KL4, KL9, KL22, KL33, KL34, KL35, KL36, KL37, KL51, KL52, KL53, and KL56were prepared by combining the corresponding aldehyde and correspondingamine in dichloroethane (DCE) in a ratio of 1.75 equivalents per aminofunction of the amine. To this solution was added at room temperaturesodium triacetoxyborohydride (NaBH(OAc)₃) (1.3 equivalents per aldehyde)and acetic acid (HOAc, 1.3 equivalents per aldehyde). The reactionmixture was stirred at ambient temperature until thin layerchromatography indicated completion of reaction. After hydrolysis with2N NaOH, the reaction mixture was extracted twice with dichloromethane(DCM). The combined organic layers were washed with saturated NaClsolution and dried over Na₂SO₄. The solvent was removed under reducedpressure and the crude product was subject to flash chromatography orreversed phase (RP) HPLC.

Amino-lipids were analyzed for purity using analytical reversed phaseHPLC. For this purpose, an XBridge C4 column (2.1×50 mm, 3.5 μm) fromWaters (Eschborn, Germany) was used. Eluent was A 0.1% TFA in water andeluent B was 0.1% TFA in 90% Acetonitrile (ACN). Elution was achieved ata column temperature of 60° C. running a gradient from 50% to 100% B in20 min at a flow rate of 0.5 mL/min. Amino-lipids were detected using anevaporative light scattering detector (PL-ELS 2100, Agilent, Waldbronn,Germany) with evaporation temperature set to 90° C., nebulizertemperature set to 40° C. and a nitrogen flow of 1 mL/sec. Identity wasestablished by electrospray ionization (ESI) mass spectrometry (MS) anddirect infusion technique.

1) Synthesis of KL22.

To a solution of Dodecanal (11.6 g, 62.8 mmol, 9.0 eq, 1.75 eq/aminefunction) and2-[4-(2-Amino-ethyl)-piperazin-1-yl]-ethyl}-ethane-1,2-diamine (1.5 g,7.0 mmol. 1.0 eq) in 100 mL dichloroethane (DCE) was added sodiumtriacetoxyborohydride (NaBH(OAc)₃) (17.3 g, 81.6 mmol, 11.7 eq) andacetic acid (HOAc) (81.6 mmol, 5.0 mL) at room temperature. The reactionmixture was stirred for 16 h at ambient temperature. After hydrolysiswith 2N NaOH the reaction mixture was extracted twice withdichloromethane (DCM). The combined organic layers were washed withsaturated NaCl-solution and dried over Na₂SO₄. The solvent was removedunder reduced pressure and the crude product was subject to flashchromatography (DCM-DCM/CH₃OH=100:6, Rf=0.05 in DCM/CH₃OH=100:1 (0.5%NEt₃)) to afford the title compound as a pale yellow oil.

ESI-MS (direct infusion): [M+H]+: 1058.5

A comparison of crude synthesis products obtained by an alternateperalkylation procedure (ring opening reaction of terminal epoxides byamines) detailed in WO2010/053572A2 and PNAS 2010, 5:1864 underscoresthe high efficiency of the chemistry disclosed herein allowing for highisolated yields (FIG. 1).

2) Synthesis of KL10.

To a solution of Dodecanal (15.1 g, 82.1 mmol, 6.0 eq) andTris-(2-aminoethyl)-amine (2.0 g, 13.7 mmol. 1.0 eq) in 100 mL DCE wasadded NaBH(OAc)₃ (26.1 g, 123.1 mmol, 9.0 eq) and HOAc (123.1 mmol, 7.0mL) at room temperature. The reaction mixture was stirred for 16 h atambient temperature. After hydrolysis with 2N NaOH the reaction mixturewas extracted twice with DCM. The combined organic layers were washedwith saturated NaCl-solution and dried over Na₂SO₄. The solvent wasremoved under reduced pressure and the crude product was subject toflash chromatography (DCM-DCM/CH₃OH=10:1-DCM/CH₃OH=5:1, R_(f) 0.01 inDCM (0.5% NEt₃)) to concentrate the title compound as a pale yellow oil.KL10 was further purified to homogeneity using RP HPLC. ESI-MS (directinfusion): [M+H]+: 986.1

3) Synthesis of KL36.

To a solution of Dodecanal (3.22 g, 17.5 mmol, 4.5 eq) andDiethylentriamine (0.40 g, 3.88 mmol. 1.0 eq) in 50 mL DCE was addedNaBH(OAc)₃ (5.56 g, 26.3 mmol, 6.75 eq) and HOAc (26.3 mmol, 1.6 mL) atroom temperature. The reaction mixture was stirred for 16 h at ambienttemperature. After hydrolysis with 2N NaOH the reaction mixture wasextracted twice with DCM. The combined organic layers were washed withsaturated NaCl-solution and dried over Na₂SO₄. The solvent was removedunder reduced pressure and the crude product was subject to flashchromatography (DCM-DCM/CH₃OH=10:1-DCM/CH₃OH=5:1, Rf=0.01 in DCM (0.5%NEt₃)) to concentrate the title compound as a pale yellow oil and toremove the excess aldehyde. The crude product was finally purified byHPLC employing a C4 reversed phase column (YMC—Pack C4, 150×20 mm, 10μm. Dienslaken, Germany). Eluent A was H₂O containing 0.1%Trifluoroacetic acid (TF A) and eluent B was 90% ACN containing 0.1%TFA. For elution at room temperature, a gradient from 70-100% Eluent Band a flow rate of 45 mL/min was used. 5 ESI-MS (direct infusion):[M+H]+: 776.7

4) Synthesis of KL37.

To a solution of octadecanal (1.20 g, 4.51 mmol, 5.5 eq) andTris-(2-aminoethyl)-amine (0.12 g, 0.82 mmol. 1.0 eq) in 50 mL DCE wasadded NaBH(OAc)₃ (1.43 g, 6.77 mmol, 6.75 eq) and HOAc (6.77 mmol, 0.4mL) at room temperature. The reaction mixture was stirred for 16 h atambient temperature. After hydrolysis with 2N NaOH, the reaction mixturewas extracted twice with DCM. The combined organic layers were washedwith saturated NaCl-solution and dried over Na₂SO₄. The solvent wasremoved under reduced pressure and the crude product was subject toflash chromatography (DCM-DCM/CH₃OH=10:1-DCM/CH₃OH=5:1, Rf=0.01 in DCM(0.5% NEW) to concentrate the title compound as a pale yellow oil and toremove the excess aldehyde. The crude product was finally purified byHPLC on a C4 reversed phase column (YMC—Pack C4, 150×20 mm, 10 μm).Eluent A was H₂O containing 0.1% Trifluoroacetic acid (TFA) and eluent Bwas 90% ACN containing 0.1% TFA. For elution at room temperature, agradient from 70-100% Eluent Band a flow rate of 45 mL/min was used.

ESI-MS (direct infusion): [M+H]+: 1386.2

C) Preparation of Selectively Alkylated Amino-Lipids.

Amino-lipids KL5, KL6, KL7, KL8, KL12, KL15, KL 16, were generated by astepwise synthetic protocol published in J Org. Chem. 1996,61:3849-3862:

The corresponding aldehyde (2.0 eq) and amine (1.0 eq) were mixed inMeOH (20 mL/1.0 mmol) at room temperature under an Argon atmosphere. Themixture was stirred at ambient temperature for 3 h, until the aldimineformation was completed. The solvent was removed under reduced pressureand the crude product was dissolved in DCE (20 mL/1.0 mmol) and treatedwith NaBH(OAc)₃ (3.0 eq) and AcOH (3.0 eq) and stirred under Argon for 3h. The reaction was quenched with 2M NaOH and the product was extractedwith EtOAc. The combined organic layers were dried over Mg₂SO₄ and thesolvent was evaporated under reduced pressure. The crude product wassubject to flash chromatography (DCM-DCM/MeOH=9:1, Rf 0.25 inDCM/MeOH=0:1 (0.5% NEt₃)) to afford the desired compound as a paleyellow oil. The final product was characterized by HPLC and massspectrometry.

D. General Synthesis Procedure for Amino-Lipids Derived from Amines andKetones.

The ketone needed for the preparation of KL32 and KL39 was prepared in 4steps according to a published synthesis route (Nature Biotechnology2010, 28:172). Other ketones are commercially available. The amine inKL23, KL30 and KL39 was made pursuing a published synthesis route (PNAS2010, 5:1864).

The amino-lipids KL23, KL24, KL25, KL26, KL27, KL28, KL30, KL32, KL39,KL43, KL49 and KL58 were prepared by combining the corresponding ketoneand the corresponding amine in DCE in a ratio of one equivalent ofketone per amine group. Subsequently, NaBH(OAc)₃ (3.0 eq) and HOAc wasadded and stirred at room temperature until thin layer chromatographyindicated completion of reaction. The reaction mixture was worked up byaddition of 2N NaOH and extraction with DCM. The organic phase was driedand the solvent removed under reduced pressure. The amino-lipids werepurified by flash column chromatography and analyzed by analyticalreversed phase HPLC and direct infusion ESI-MS.

1) Preparation of KL25.

To a solution of Heptacosan-14-one (11.0 g, 27.9 mmol, 2.0 eq, 1.0eq/amine function) and Triethylenetetraamine (2.03 g, 13.9 mmol. 1.0 eq)in 200 mL DCE was added NaBH(OAc)₃ (8.90 g, 41.9 mmol, 3.0 eq) and HOAc(41.9 mmol, 2.5 mL) at room temperature. The reaction mixture wasstirred for 72 h at ambient temperature. After hydrolysis with 2N NaOHthe reaction mixture was extracted twice with DCM. The combined organiclayers were washed with saturated NaCl solution and dried over Na₂S0₄.The solvent was removed under reduced pressure and the crude product wassubject to flash chromatography (DCM-DCM/CH₃OH=10:1-DCM/CH₃OH 5:1,R_(f)=0.3 in DCM/CH₃OH=4:1 (0.1% aq. NH₃)) to afford the title compoundas a pale yellow wax.

ESI-MS (direct infusion): [M+H]+: 904.0

Example 2 siRNA Synthesis

siRNAs were synthesized by standard solid phase RNA oligomerizationusing the phosphoramidite technology. Depending on the scale either anABI 394 synthesizer (Applied Biosystems) or an Äkta oligopilot 100 (GEHealthcare, Freiburg, Germany) was used. In order to increase siRNAstability and abrogate immune responses, 2′-O-methyl modifiednucleotides were placed within certain positions in the siRNA duplex.Ancillary synthesis reagents, RNA and 2′-O-Methyl RNA phosphoramiditeswere obtained from SAFC Proligo (Hamburg, Germany). Specifically,5′-O-(4,4′-dimethoxytrityl)-3′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite monomers of uridine (U), 4-N-acetylcytidine (C^(Ac)),6-N-benzoyladenosine (A^(bz)) and 2-N-isobutyrlguanosine (G^(iBu)) with2′-O-t-butyldimethylsilyl were used to build the oligomers sequence.2′-O-Methyl modifications were introduced employing the correspondingphosphoramidites carrying the same nucleobase protecting groups as theregular RNA building blocks. Coupling time for all phosphoramidites (100mM in Acetonitrile) was 6 min employing 5-Ethylthio-1H-tetrazole (ETT)as activator (0.5 M in Acetonitrile). Phosphorothioate linkages wereintroduced using 50 mM3-((Dimethylamino-methylidene)amino)-3H-1,2,4-dithiazole-3-thione (DDTT,AM Chemicals, Oceanside, Calif., USA) in a 1:1 (v/v) mixture of pyridineand Acetonitrile. Upon completion of the solid phase synthesisoligoribonucleotides were cleaved from the solid support and deprotectedusing slight modification of published methods (Wincott F. et al.“Synthesis, deprotection, analysis and purification of RNA andribozymes.” Nucleic Acids Res 1995, 23:2677-2684).

Crude oligomers were purified by anionic exchange HPLC using a columnpacked with Source Q15 (GE Healthcare) and an Äkta Explorer system (GEHealthcare). Buffer A was 10 mM sodium perchlorate, 20 mM Tris, 1 mMEDTA, pH 7.4 (Fluka, Buchs, Switzerland) and contained 20% Acetonitrile.Buffer B was the same as buffer A with the exception of 500 mM sodiumperchlorate. A gradient of 22% B to 42% B within 32 column volumes (CV)was employed. UV traces at 280 nm were recorded. Appropriate fractionswere pooled and precipitated with 3M NaOAc, pH 5.2 and 70% ethanol.Finally, the pellet was washed with 70% ethanol.

Isolated RNAs were shown to be at least 85% pure by analytical stronganion exchange chromatography. Identity of the RNA single strands wasconfirmed by LC-ESI-MS.

siRNAs were prepared by combining equimolar amounts of the complementaryRNA strands in sodium citrate buffer (10 mM Na-Citrate, 30 mM NaCl, pH6), heating to 70° C. for 5 min and slow cooling to room temperatureover a time period of 2 h. siRNAs were further characterized bycapillary gel electrophoresis and were stored frozen until use.

siRNA sequences are listed in Table 2. As indicated in the table, thesesiRNAs are directed against gene targets that are exclusively expressedin certain cells in the liver. In certain embodiments the individualsiRNAs were employed in the inventive formulations. In certain otherembodiments a mixture of all the indicated siRNAs were incorporated intothe inventive formulations to address siRNA delivery to other cell typesthan hepatocytes. Those additional cell types are listed in Table 2 aswell.

TABLE 2 siRNAs employed in LNPs of the present invention. Duplex-IDssRNA ID Sequence 5′-3′ type Target Cell type 1/2 1GcAAAGGcGuGccAAcucAdTsdT s Factor VII hepatocytes 1/2 2UGAGUUGGcACGCCUUUGCdTsdT as 3/4 3 AcGucuAuAucAuGGccGAdTsdT s EGFPubiquitous 3/4 4 UCGGCcAUGAuAuAGACGUdTsdT as 7/8 7cuuuucucGuGAcAAGAAGdTsdT s CLEC4F Kupffer cells 7/8 8CUUCUUGUcACGAGAAAAGdTsdT as  9/10 9 GGucucAAGccAcucGuuudTsdT s RELNStellate cells  9/10 10 AAACGAGUGGCUUGAGACCdTsdT as 11/12 11GAAGAuGcAGuGAuuuAcAdTsdT s TEK endothelial 11/12 12UGuAAAUcACUGcAUCUUCdTsdT as cells 13/14 13 GcGcAGAAuucAucucuucdTsdT sCD68 Macrophages 13/14 14 GAAGAGAUGAAUUCUGCGCdTsdT as 15/16 15cuGGcuGAAuuucAGAGcAdTsdT s CD45 Leukocytes 15/16 16UGCUCUGAAAUUcAGCcAGdTsdT as 5/6 5 AAcGAGAAGcGcGAucAcAdTsdT s EGFPUbiquitous 5/6 6 UGUGAUCGCGCUUCUCGUUdTsdT as 17/18 17agAuGGAuAuAcucAAuuAdTsdT s Clec7a Macrophages 17/18 18uAAUUGAGuAuAUCcAUCUdTsdT as Key: Upper case letters A, C, G, U representRNA nucleotides; lower case letters a, c, g, u, are 2′-O-Methylnucleotides. A phosphorothioate linkage is symbolized with a lower case“s”. dT is deoxythimidine.

Example 3 Lipid Nanoparticles

A. siRNA Lipid Nanoparticle Preparation.

Helper lipids were purchased from Avanti Polar Lipids (Alabaster, Ala.,USA). PEGylated lipids were obtained from NOF (Bouwelven, Belgium).Small molecules such as cholesterol were purchased from Sigma-Aldrich(Taufkirchen, Germany).

Lipid nanoparticles containing siRNAs as described in the section belowwere compared against a standard formulation containing the lipid2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (XTC2) discovered byTekmira Pharmaceuticals. XTC2 was synthesized according to publishedprocedures (Nature Biotechnology, 2010, 28:172). The correspondingstandard formulation was prepared, unless otherwise stated, according toa published composition (PNAS, 2010, 107:1854). For this purpose, stocksolutions of 1,2-distearoyl-3-phosphatidylcholine (DSPC, 10%), XTC2(50%), cholesterol (38.5%), andα-[3′-(1,2-dimyristoyl-3-propanoxy)-carboxamide-propyl]-ω-methoxy-polyoxyethylene(PEG-c-DOMG 1.5%) were prepared at concentrations of 50 mM in ethanol.

The inventive lipid nanoparticle formulations of the present inventioncontain the amino-lipids disclosed herein instead of XTC2. Initially,the other components were kept unchanged.

siRNA stock solutions at a concentration of 10-20 mg/mL in 10 mM sodiumcitrate buffer, 30 mM NaCl, pH 6 were diluted in 50 mM citrate buffer,pH 4 to the desired total siRNA concentration (˜1 mg/mL).

siRNA lipid nanoparticles were manufactured at a total lipid to siRNAmass ratio of 7 by combining the lipid solution in ethanol with thebuffered siRNA solution in a mixing tee (e.g. CMIXPK, VICI AGInternational, Schenkon, Switzerland) by using either a Harvard Pump 33Syringe Pump (Harvard Apparatus Holliston, Mass.) or for larger batches(>15 mL) an Äkta 900 HPLC Pump (GE Healthcare Bio-Sciences Corp.,Piscataway, N.J.). Flow rates ranged from (17 mL/min to 67 mL/min forthe siRNA solution and from 8 mL/min to 33 mL/min for the lipidsolution.

Subsequent to the initial testing in mice efficacious siRNA lipidnanoparticle formulations were further optimized. Formulation variationswere generated by variation of the compositions. Differences betweenformulations reflect differences in the lipid species or differences inthe molar percentages of the lipid components, or differences in theratio between positively and negatively charged components of theformulation.

The primary product, i.e. the product resulting by combining the twoinput solutions, was dialyzed 2× against phosphate buffered saline(PBS), pH 7.4 at volumes 100× of that of the primary product usingSpectra/Por dialysis tubing (Spectrum Europe B. V., Breda, TheNetherlands) with a MWCO of 100 or 250 kDa (CE, or PVDF membrane) orusing Slide-A-Lyzer cassettes (Thermo Fisher Scientific Inc. Rockford,Ill.) with a MWCO of 10 kD (RC membrane).

If desired siRNA lipid nanoparticles were concentrated at a 4° C. byusing Vivaspin 20 centrifugation tubes (Sartorius AG, Gottingen,Germany) with a MWCO of 50 kD at 700 g using a table-top centrifuge.

The lipid nanoparticle suspension was filtered through a Filtropur S 0.2filter with a pore size of 0.2 μm (Sarstedt, Numbrecht, Germany) andfilled into glass vials with a crimp closure.

B. siRNA Lipid Nanoparticle Characterization.

To determine the siRNA concentration, formulations were diluted to atheoretical siRNA concentration of approximately 0.02 mg/mL in phosphatebuffered saline (PBS). A volume of 100 μL of the diluted formulation wasadded to 900 μL of a 4:1 (vol/vol) mixture of methanol and chloroform.After vigorous mixing for 1 min, the absorbance spectrum of the solutionwas recorded at wavelengths between 230 nm and 330 nm on a DU 800spectrophotometer (Beckman Coulter, Beckman Coulter, Inc., Brea,Calif.). The siRNA concentration in the liposomal formulation wasdetermined based on the extinction coefficient of the siRNA used in theformulation. If extinction coefficient was not known, an average valueof 22 OD/mg was used. The siRNA concentration was calculated based onthe difference between the absorbance maximum at a wavelength of ˜260 nmand the baseline value at a wavelength of 330 nm.

To determine the mean size of siRNA lipid nanoparticles, formulationswere diluted in PBS to a concentration of approximately 0.05 mg/mL siRNAin a disposable polystyrene cuvette. The mean particle size wasdetermined by using a Zetasizer Nano ZS (Malvern Instruments Ltd,Malvern, Worcestershire, UK).

To determine the zeta potential of siRNA lipid nanoparticles,formulations were diluted in PBS, pH 7.4 and in citrate buffer, pH 4 toa concentration of approximately 0.01 mg/mL siRNA in a disposable zetacell (DTS1060C, Malvern Instruments Ltd, Malvern, Worcestershire, UK).The zeta potential was determined by using a Zetasizer Nano ZS (MalvernInstruments Ltd, Malvern, Worcestershire, UK).

To determine the percentage of siRNA entrapped in lipid nanoparticlesthe Quant-iT™ RiboGreen® RNA assay (Invitrogen Corporation Carlsbad,Calif.) was used according to the manufacturer's instructions. In brief,samples were diluted to a concentration of approximately 5 μg/mL in TEbuffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5). Volumes of 50 μL of thediluted samples were transferred to a polystyrene 96 well plate. To thesamples, either 50 μL of TE buffer or 50 μL of a 2% Triton X-100solution was added. The plate was incubated at 35° C. for 15 min. TheRiboGreen reagent was diluted 1:100 in TE buffer and a volume of 100 μLof was added to each well. The fluorescence intensity in each well wasdetermined using a fluorescence plate reader (Wallac Victor 1420Multilabel Counter; Perkin Elmer, Waltham, Mass.) at an excitationwavelength of ˜480 nm and an emission wavelength of ˜520 nm. Thefluorescence values of the reagent blank were subtracted from that ofeach of the samples and siRNA concentrations were determined based on astandard curve of fluorescence intensities versus RNA concentrations.The percentage of free siRNA was determined by dividing the fluorescenceintensity of the intact sample (without addition of Triton X-100) by thefluorescence value of the disrupted sample (with addition of TritonX-100).

Example 4 Animal Experiments

Mice (strain C57BL/6) were obtained from Charles River (Sulzfeld,Germany) or were bred in house (EGFP transgenic mice in C57Bl/6background) and were between 6 and 8 weeks 30 old at the time of theexperiments. Intravenously administered LNPs were injected by infusionof 200 μL into the tail vein. LNPs administered to the lung wereorotracheally instilled by applying 50 μL into the pharynx of isofluraneanaesthetized mice and making mice breathe in the LNP solution whileblocking their obligate nose breathing. 48 h post administration, micewere anaesthetized by CO₂ inhalation and sacrificed by cervicaldislocation. Blood was collected during the experiment by submandibularvein bleed or—after sacrificing the animals—by cardiac puncture andserum isolated with serum separation tubes (Greiner Bio-One,Frickenhausen, Germany). Factor VII protein levels were analyzed by achromogenic assay (see below). For preparation of bronchoalveolar lavage(BAL) fluid, the lungs were flushed 3× with 1 ml PBS via anintratracheally inserted canula and cells were pelleted bycentrifugation. For quantitation of mRNA levels, organs were harvestedand organ homogenates were prepared. Tissues were snap frozen in liquidnitrogen and powdered with mortar and pestle on dry ice. 30-50 mg oftissue was transferred to a chilled 1.5 mL reaction tube. 1 mL LysisMixture (Epicenter Biotechnologies, Madison, USA) and 3.3 μL ProteinaseK (50 μg/μL) (Epicenter Biotechnologies, Madison, USA) was added andtissues were lysed by sonication for several seconds using a sonicator(HD2070, Bandelin, Berlin, Germany) and digested with Proteinase K for15 min at 65° C. in a thermomixer (Thermomixer comfort, Eppendorf,Hamburg, Germany). BAL lysates were obtained by resuspending BAL cellsin 200 μl, Lysis Mixture, followed by incubation at 53° C. for 30 min.Lysates were stored at −80° C. until analysis. For mRNA analyses,lysates were thawed and mRNA levels were measured using eitherQuantiGene 1.0 or Quantigene 2.0 branched DNA (bDNA) Assay Kit(Panomics, Fremont, Calif., USA, Cat-No: QG0004) according to themanufacturer's recommendations. In order to assess the FVII, EGFP,Clec4f, RELN, TEK, CD45, CD68, Clec7a and GAPDH mRNA content, thefollowing probe sets were employed:

TABLE 3 Quantigene 1.0 probe sets. SEQ Oligo Name Sequence 5′ → 3′ID No. mRNA mGAP 2001 gagagcaatgccagccccTTTTTctcttggaaagaaagt 19mouse GAPDH mGAP 2002 ggtccagggtttcttactccttgTTTTTctcttggaaagaaagt 20mouse GAPDH mGAP 2003 ccctaggcccctcctgttattTTTTTctcttggaaagaaagt 21mouse GAPDH mGAP 2004 tgcagcgaactttattgatggTTTTTctcttggaaagaaagt 22mouse GAPDH mGAP 2005 gcacgtcagatccacgacgTTTTTaggcataggacccgtgtct 23mouse GAPDH mGAP 2006 ggcaggtttctccaggcgTTTTTaggcataggacccgtgtct 24mouse GAPDH mGAP 2007 gccctcagatgcctgcttcaTTTTTaggcataggacccgtgtct 25mouse GAPDH mGAP 2008 gccgtattcattgtcataccaggTTTTTaggcataggacccgtgtct 26mouse GAPDH mGAP 2009 gtccaccaccctgttgctgtaTTTTTaggcataggacccgtgtct 27mouse GAPDH mGAP 2010 aattgtgagggagatgctcagtTTTTTaggcataggacccgtgtct 28mouse GAPDH mGAP 2011 ccaccttcttgatgtcatcatactt 29 mouse GAPDH mGAP 2012cccaagatgcccttcagtgg 30 mouse GAPDH mGAP 2013 gagacaacctggtcctcagtgtag31 mouse GAPDH mGAP 2014 ggagttgctgttgaagtcgcag 32 mouse GAPDH mGAP 2015ggcatcgaaggtggaagagtg 33 mouse GAPDH mGAP 2016 aaatgagcttgacaaagttgtcatt34 mouse GAPDH mGAP 2017 gaggccatgtaggccatgag 35 mouse GAPDH mGAP 2018gtccttgctggggtgggt 36 mouse GAPDH mGAP 2019 tagggcctctcttgctcagt 37mouse GAPDH mGAP 2020 gttgggggccgagttggga 39 mouse GAPDH mGAP 2021atgggggtctgggatgga 39 mouse GAPDH mGAP 2022 tattcaagagagtagggagggct 40mouse GAPDH mmFak7 001 gagaagcagcagcccatgcTTTTTctcttggaaagaaagt 41mouse Factor VII mmFak7 002 tggagctggagcagaaagcaTTTTTctcttggaaagaaagt 42mouse Factor VII mmFak7 003 tgcttcctcctgggttatgaaaTTTTTctcttggaaagaaagt43 mouse Factor VII mmFak7 004 ccgggccaaagctcctccTTTTTctcttggaaagaaagt44 mouse Factor VII mmFak7 005 cttgaagatctcccgggccTTTTTctcttggaaagaaagt45 mouse Factor VII mmFak7 006ctgcttggtcctctcagggctTTTTTctcttggaaagaaagt 46 mouse Factor VIImmFak7 007 actgcagtccctagaggtcccTTTTTaggcataggacccgtgtct 47mouse Factor VII mmFak7 008tttgcctgtgtaggacaccatgTTTTTaggcataggacccgtgtct 48 mouse Factor VIImmFak7 009 tcctcaaaggagcactgttccTTTTTaggcataggacccgtgtct 49mouse Factor VII mmFak7 010cccccatcactgtaaacaatccagaaTTTTTaggcataggacccgtgtct 50 mouse Factor VIImmFak7 011 tggattcgaggcacactggtTTTTTaggcataggacccgtgtct 51mouse Factor VII mmFak7 012tggcaggtacctacgttctgacaTTTTTaggcataggacccgtgtct 52 mouse Factor VIImmFak7 013 cttgcttttctcacagttccgaTTTTTaggcataggacccgtgtct 53mouse Factor VII mmFak7 014 aggagtgagttggcacgcc 54 mouse Factor VIImmFak7 015 tcattgcactctctctccagagag 55 mouse Factor VII mmFak7 016gcagacgtaagacttgagatgatcc 56 mouse Factor VII mmFak7 017ccctcaaagtctaggaggcagaa 57 mouse Factor VII mmFak7 018ttgcacagatcagctgctcatt 58 mouse Factor VII EGFP 001ggcacgggcagcttgcTTTTTctcttggaaagaaagt 59 EGFP EGFP 002ggtagcggctgaagcactgTTTTTctcttggaaagaaagt 60 EGFP EGFP 003cctggacgtagccttcgggTTTTTctcttggaaagaaagt 61 EGFP EGFP 004ccttgaagaagatggtgcgctTTTTTctcttggaaagaaagt 62 EGFP EGFP005cgaacttcacctcggcgcTTTTTctcttggaaagaaagt 63 EGFP EGFP006ccttcagctcgatgcggtTTTTTctcttggaaagaaagt 64 EGFP EGFP 007gtcacgagggtgggccagTTTTTaggcataggacccgtgtct 65 EGFP EGFP 008cacgccgtagtcagggtgTTTTTaggcataggacccgtgtct 66 EGFP EGFP 009gtgctgcttcatgtggtcggTTTTTaggcataggacccgtgtct 67 EGFP EGFP 010tcaccagggtgtcgccctTTTTTaggcataggacccgtgtct 68 EGFP EGFP 011cggtggtgcagatgaacttca 69 EGFP EGFP 012 catggcgacttgaagaagtc 70 EGFPEGFP 013 cgtcctccttgaagtcgatgc 71 EGFP mmClec4f 001ggtcccttctcagggtctgtaaTTTTTctcttggaaagaaagt 72 mouse Clec4f mmClec4f 002tctgtcttggccctctgaagatTTTTTctcttggaaagaaagt 73 mouse Clec4f mmClec4f 003ccccaggcgattctgctcTTTTTctcttggaaagaaagt 74 mouse Clec4f mmClec4f 004tctgctcctgcttctgtgcagTTTTTctcttggaaaeaaagt 75 mouse Clec4f mmClec4f 005cagaacttctcagcctcccgTTTTTctcttggaaagaaagt 76 mouse Clec4f mmClec4f 006tgcgctccctgggacgtaTTTTTctcttggaaagaaagt 77 mouse Clec4f mmClec4f 007gacttcaaagctgagacatcactcaTTTTTaggcataggacccgtgtct 78 mouse Clec4fmmClec4f 008 gatctgccttcaaactctgcatcTTTTTaggcataggacccgtgtct 79mouse Clec4f mmClec4f 009 ggctttggtcgcctgcaTTTTTaggcataggacccgtgtct 80mouse Clec4f mmClec4f 010 ttccagttctgcgcgatcaTTTTTaggcataggacccgtgtct 81mouse Clec4f mmClec4f 011 agaggtcaccgaagccaggTTTTTaggcataggacccgtgtct 82mouse Clec4f mmClec4f 012 tgctctgtagcatctggacattg 83 mouse Clec4fmmClec4f 013 cccctgaatcttggcagtgag 84 mouse Clec4f mmClec4f 014ccacagcttcctgcagggc 85 mouse Clec4f mmClec4f 015gctggagaacctgattctgagtct 86 mouse Clec4f mmClec4f 016aaaagtaataaaagtttccattgaagtac 87 mouse Clec4f mmClec4f 017ccacggcttcttgtcacgag 88 mouse Clec4f mmReln 001cagagatcttgaactgcatgatccTTTTTctcttggaaagaaagt 89 mouse Reln mmReln 002tcggcgggtaagcactgaTTTTTctcttggaaagaaagt 90 mouse Reln mmReln 003cgcttccagaacactttgggTTTTTctcttggaaagaaagt 91 mouse Reln mmReln 004ttcaggaagcgggtaggtgaTTTTTctcttggaaagaaagt 92 mouse Reln mmReln 005gtccatcatggctgccacaTTTTTaggcataggacccgtgtct 93 mouse Reln mmReln 006tcatgagtcactgcatacacctctcTTTTTaggcataggacccgtgtct 94 mouse RelnmmReln 007 ttcaggcactttgcatccaaTTTTTaggcataggacccgtgtct 95 mouse RelnmmReln 008 tgaatttgattctgggcaattttTTTTTaggcataggacccgtgtct 96 mouse RelnmmReln 009 gggactaaataactccagctcacgTTTTTaggcataggacccgtgtct 97mouse Reln mmReln 010 tccttttccacccttcagttgTTTTTaggcataggacccgtgtct 98mouse Reln mmReln 011 ttacaggattccccgttaagctTTTTTaggcataggacccgtgtct 99mouse Reln mmReln 012 gggtcacagatacactgttcctttTTTTTaggcataggacccgtgtct100 mouse Reln mmReln 013 agtcacagaatctttccactgtacag 101 mouse RelnmmReln 014 gagcatgacaccatctggcg 102 mouse Reln mmReln 015agttctcagtgggcgtcagg 103 mouse Reln mmReln 016 ccaaagtcagtagaaaactgcacg104 mouse Reln mmReln 017 ggaagaacacagacggttgagaaa 105 mouse RelnmmReln 018 tctgagtacttttggtagaacctaaatc 106 mouse Reln mmTek 001tgagtccctgggaagctttcaTTTTTctcttggaaagaaagt 107 mouse Tek mmTek 002acttccccagatctccccatTTTTTctcttggaaagaaagt 108 mouse Tek mmTek 003taagccggctaaagagtccatTTTTTctcttggaaagaaagt 109 mouse Tek mmTek 004aatgcaggtgagggatgtttTTTTTctcttggaaagaaagt 110 mouse Tek mmTek 005tcctatggtgatgggctcatggTTTTTctcttggaaagaaagt 111 mouse Tek mmTek 006ccgctcgcatggtccacgTTTTTaggcataggacccgtgtct 112 mouse Tek mmTek 007acaactcacaactttgcgacttcTTTTTaggcataggacccgtgtct 113 mouse Tek mmTek 008ccagcgtccacagatgagcaTTTTTaggcataggacccgtgtct 114 mouse Tek mmTek 009agcaagctgactccacagagaacTTTTTaggcataggacccgtgtct 115 mouse Tek mmTek 010gcgccttctactactccataaaggTTTTTaggcataggacccgtgtct 116 mouse Tek mmTek 011cggcatcagacacaagaggtaggTTTTTaggcataggacccgtgtct 117 mouse Tek mmTek 012gggtgccacccagaggcTTTTTaggcataggacccgtgtct 118 mouse Tek mmTek 013gcaaggagaaacaccacagaag 119 mouse Tek mmTek 014 cgctcttgtttacaagttggcg120 mouse Tek mmTek 015 gaattgatcaagatcaggtccatg 121 mouse Tek

TABLE 4 Quantigene 2.0 probe sets. SEQ Oligo Name Sequence 5′ → 3′ID No. mRNA Q2 mCD45 1 tgacgagttttacaccgcgatTTTTTgaagttaccgtttt 122mouse CD45 Q2 mCD45 2 aatctgtctgcacatttataacattttTTTTTctgagtcaaagcat 123mouse CD45 Q2 mCD45 3 ggcgtttctggaatccccaTTTTTctcttggaagaaagt 124mouse CD45 Q2 mCD45 4 tggatccccacaactaggcttaTTTTTgaagttaccgtttt 125mouse CD45 Q2 mCD45 5 agagactaccgctttttcttgcagcTTTTTctgagtcaaagcat 123mouse CD45 Q2 mCD45 6 gtttagatacaggctcaggccaTTTTTctcttggaaagaaagt 127mouse CD45 Q2 mCD45 7 tggggtttagatgcagactcagTTTTTgaagttaccgtttt 128mouse CD45 Q2 mCD45 8 attgttcttatagcataaaacatatccaTTTTTctgagtcaaagcat129 mouse CD45 Q2 mCD45 9 taggcaaacttttacatttttctgaTTTTTctcttggaaagaaagt130 mouse CD45 Q2 mCD45 10 ccacctcaaaactggtcacattatTTTTTgaagttaccgtttt131 mouse CD45 Q2 mCD45 11tcatagtatttataaggtttcaagctttTTTTTctgagtcaaagcat 132 mouse CD45Q2 mCD45 12 ttgacataggcaagtagggacact 133 mouse CD45 Q2 mCD45 13cccatttctttgaatcttcccaTTTTTctcttggaaagaaagt 134 mouse CD45 Q2 mCD45 14tgaaaattgcacttctcagcagtTTTTTgaagttaccgtttt 135 mouse CD45 Q2 mCD45 15ccggacgatctgcttttgtgTTTTTctgagtcaaagcat 136 mouse CD45 Q2 mCD45 16ggttttcattccattgaccttgtTTTTTctcttggaaagaaagt 137 mouse CD45 Q2 mCD45 17ttgtctgtcggccgggaTTTTTgaagttaccgtttt 138 mouse CD45 Q2 mCD45 18ggaggaccacatgtaacatttatactaTTTTTctgagtcaaagcat 139 mouse CD45Q2 mCD45 19 ggttttagggccattagtttcataaTTTTTctcttggaaagaaagt 140mouse CD45 mGAPDH QG2 1 cgaggctggcactgcacaaTTTTTctcttggaaagaaagt 141mouse GAPDH mGAPDH QG2 2 cttcaccattttgtctacgggaTTTTTgaagttaccgtttt 142mouse GAPDH mGAPDH QG2 3 ccaaatccgttcacaccgacTTTTTctgagtcaaagcat 143mouse GAPDH mGAPDH QG2 4 ccaggcgcccaatacggTTTTTctcttggaaagaaagt 144mouse GAPDH mGAPDH QG2 5 caaatggcagccctggtgaTTTTTgaagttaccgtttt 145mouse GAPDH mGAPDH QG2 6 aacaatctccactttgccactgTTTTTctgagtcaaagcat 146mouse GAPDH mGAPDH QG2 7 tgaaggggtcgttgatggc 147 mouse GAPDHmGAPDH QG2 8 catgtagaccatgtagttgaggtcaa 148 mouse GAPDH mGAPDH QG2 9ccgtgagtggagtcatactggaaTTTTTctcttggaaagaaagt 149 mouse GAPDHmGAPDH QG2 10 ttgactgtgccgttgaatttgTTTTTctcttggaaagaaagt 150 mouse GAPDHmGAPDH QG2 11 agcttcccattctcggccTTTTTgaagttaccgtttt 151 mouse GAPDHmGAPDH QG2 12 gggcttcccgttgatgacaTTTTTctgagtcaaagcat 152 mouse GAPDHmGAPDH QG2 13 cgctcctggaagatggtgatTTTTTctcttggaaagaaagt 153 mouse GAPDHmGAPDH QG2 14 cccatttgatgttagtggggtct 154 mouse GAPDH mGAPDH QG2 15atactcagcaccggcctcacTTTTTctcttggaaagaaagt 155 mouse GAPDH QG2 mCD68 1ctgggagccgttggccTTTTTctcttggaaagaaagt 156 mouse CD68 QG2 mCD68 2ggcttggagctgaacacaaggTTTTTgaagttaccgtttt 157 mouse CD68 0G2 mCD68 3ggtataggattcggatttgaatttgTTTTTctgagtcaaagcat 158 mouse CD68 QG2 mCD68 4acctttcttccaccctgaattgTTTTTctcttggaaagaaagt 159 mouse CD68 QG2 mCD68 5tctttaagccccactttagctttTTTTTgaagttaccgtttt 160 mouse CD68 0G2 mCD68 6acagatatgccccaagcccTTTTTctgagtcaaagcat 161 mouse CD68 QG2 mCD68 7cttggttttgttgggattcaaaTTTTTgaagttaccgtttt 162 mouse CD68 0G2 mCD68 8ccgtcacaacctccctggacTTTTTctgagtcaaagcat 163 mouse CD68 QQ2 mCD68 9agagacaggtggggatgggtaTTTTTctcttggaaagaaagt 164 mouse CD68 QG2 mCD68 10ggtaagctgtccataaggaaatgagTTTTTgaagttaccgtttt 165 mouse CD68 QG2 mCD68 11tgtaggtcctgtttgaatccaaaTTTTTctgagtcaaagcat 166 mouse CD68 QG2 mCD68 12ggtagactgtactcgggctctgaTTTTTctcttggaaagaaagt 167 mouse CD68 QG2 mCD68 13tccaccgccatgtagtccaTTTTTgaagttaccgtttt 168 mouse CD68 QG2 mCD68 14cctgtgggaaggacacattgtatTTTTTctgagtcaaagcat 169 mouse CD68 QG2 mCD68 15ccatgaatgtccactgtgctgTTTTTgaagttaccgtttt 170 mouse CD68 QG2 mCD68 16tctcgaagagatgaattctgcgTTTTTctgagtcaaagcat 171 mouse CD68 QG2 mCD68 17cccaagggagcttggagcTTTTTctcttggaaagaaagt 172 mouse CD68 QG2 mCD68 18tttccacagcagaagctttgg 173 mouse CD68 QG2 mCD68 19ctggagaaagaactatgcttgca 174 mouse CD68 QG2 mCD68 20agagagcaggtcaaggtgaacagTTTTTctcttggaaagaaagt 175 mouse CD68 QG2 eGFP 1ggctgaagcactgcacgcTTTTTgaagttaccgtttt 176 EGFP QG2 eGFP 2gaagtcgatgcccttcagctTTTTTctgagtcaaa.e;cat 177 EGFP QG2 eGFP 3ggatgttgccgtcctccttTTTTTgaagttaccgtttt 178 EGFP QG2 eGFP 4tactccagcttgtgccccaTTTTTctgagtcaaagcat 179 EGFP QG2 eGFP 5agacgttgtggctgttgtagttgTTTTTgaagttaccgtttt 180 EGFP 0G2 eGFP 6tctgcttgtcggccatgatatTTTTTctgagtcaaagcat 181 EGFP QG2 eGFP 7aagttcaccttgatgccgttctTTTTTgaagttaccgtttt 182 EGFP QG2 eGFP 8cgatgttgtggcggatcttgTTTTTctgagtcaaagcat 183 EGFP QG2 eGFP 9ctgcacgctgccgtcctTTTTTctcttggaaagaaagt 184 EGFP 0G2 eGFP 10gctggtagtggtcggcgag 185 EGFP QG2 eGFP 11 cgccgatgggggtgttct 186 EGFPQG2 EGFP 12 cttcatgtggtcggggtagcTTTTTctgagtcaaagcat 187 EGFP QG2 EGFP 13gcagcacggggccgt 188 EGFP QG2 EGFP 14 caggtagtggttgtcgggca 189 EGFPQG2 EGFP 15 agggcggactgggtgctTTTTTctcttggaaagaaagt 190 EGFP QG2 EGFP 16tctcgttggggtctttgctc 191 EGFP QG2 EGFP 17aggaccatgtgatcgcgctTTTTTctcttggaaagaaagt 192 EGFP QG2 EGFP 18gcggtcacgaactccagcTTTTTctcttggaaagaaagt 193 EGFP QG2 EGFP 19cggacttgaagaagtcgtgctg 194 EGFP QG2 EGFP 20cgtagccttcgggcatggTTTTTctcttggaaagaaagt 195 EGFP QG2 EGFP 21aagatggtgcgctcctggaTTTTTgaagttaccgtttt 196 EGFP QG2 EGFP 22agttgccgtcgtccttgaagTTTTTctgagtcaaagcat 197 EGFP QG2 EGFP 23tcggcgcgggtcttgt 198 EGFP QG2 EGFP 24gtcgccctcgaacttcaccTTTTTctcttggaaagaaagt 199 EGFP QG2 EGFP 25cgatgcggttcaccagggtTTTTTgaagttaccgtttt 200 EGFP QG2 mClec7a 1tttctctgatcccctgggcTTTTTgaagttaccgtttt 201 mouse Clec7a QG2 mClec7a 2gaagatggagcctggcttccTTTTTctgagtcaaagcat 202 mouse Clec7a QG2 mClec7a 3gcaatgggcctccaaggtTTTTTctcttggaaagaaagt 203 mouse Clec7a QG2 mClec7a 4gcacaggattcctaaacccactTTTTTgaagttaccgtttt 204 mouse Clec7a 0G2 mClec7a 5gcagcaaccactactaccacaaaTTTTTctgagtcaaagcat 205 mouse Clec7aQG2 mClec7a 6 tgctagggcacccagcactTTTTTctcttggaaagaaagt 206 mouse Clec7aQG2 mClec7a 7 cctgaattgtgtcgccaaaaTTTTTgaagttaccgtttt 207 mouse Clec7aQG2 mClec7a 8 ttgtctttctcctctggatttctcTTTTTctgagtcaaagcat 208mouse Clec7a QG2 mClec7a 9tggttctctttatttcttgataggaagTTTTTctcttggaaagaaagt 209 mouse Clec7aQG2 mClec7a 10 ctaaagatgattctgtgggcttgTTTTTgaagttaccgtttt 210mouse Clec7a QG2 mClec7a 11 ggagggagccaccttctcatTTTTTctgagtcaaagcat 211mouse Clec7a QG2 mClec7a 12 ctcctgtagtttgggatgccttTTTTTctcttggaaagaaagt212 mouse Clec7a QG2 mClec7a 13ggaaggcaaggctgagaaaaacTTTTTgaagttaccgtttt 213 mouse Clec7aQG2 mClec7a 14 cttcccatgcatgatccaattaTTTTTctgagtcaaagcat 214mouse Clec7a QG2 mClcc7a 15cctgagaagctaaataggtaacagctTTTTTctcttggaaagaaagt 215 mouse Clec7aQG2 mClec7a 16 tctcttacttccataccaggaatttTTTTTgaagttaccgtttt 216mouse Clec7a QG2 mClec7a 17 gcacctagctgggagcagtgTTTTTctgagtcaaagcat 217mouse Clec7a 0G2 mClec7a 18 ttttgagttgtctatcttcagtagatga 218mouse Clec7a QG2 mClec7a 19 tggctttcaatgaactcaaattcTTTTTctcttggaaagaaagt219 mouse Clec7a 0G2 mClec7a 20gcattaatacggtgagacgatgttTTTTTgaagttaccgtttt 220 mouse Clec7aQG2 mClec7a 21 cgggaaaggcctatccaaaaTTTTTctgagtcaaagcat 221 mouse Clec7aQG2 mClec7a 22 catggcccttcactctgattgTTTTTctcttggaaagaaagt 222mouse Clec7a QG2 mClec7a 23 gctgatccatcctcccagaacTTTTTctcttggaaagaaagt223 mouse Clec7a QG2 mClec7a 24ttgaaacgattggggaagaatTTTTTctcttggaaagaaagt 224 mouse Clec7aQG2 mClec7a 25 cctggggagctgtatttctgacTTTTTgaagttaccgtttt 225mouse Clec7a QG2 mClec7a 26 catacacaattgtgcagtaagctttTTTTTctgagtcaaagcat226 mouse Clec7a

The bDNA assay was performed using 20 μL lysate and the correspondinggene specific probe sets. For normalization purposes GAPDH mRNAexpression was analyzed using 40 μL lysate and Rattus norvegicus probesets shown to be cross-react with mice (sequences of probe sets seeabove). As assay readout the chemiluminescence signal was measured in aVictor 2 Light luminescence counter (Perkin Elmer, Wiesbaden, Germany)as relative light units (RLU). The signal for the corresponding mRNA wasdivided by the signal for GAPDH mRNA from the same lysate. Values arereported as mRNA expression normalized to GAPDH.

For measurement of FVII activity, plasma samples from mice were preparedby collecting blood (9 volumes) by submandibular bleeding intomicrocentrifuge tubes containing 0.109 mol/L sodium citrateanticoagulant (1 volume) following standard procedures. FVII activity inplasma was measured with a chromogenic method using a BIOPHEN VII kit(Hyphen BioMed/Aniara, Mason, Ohio) following manufacturer'srecommendations. Absorbance of colorimetric development was measuredusing a Tecan Safire2 microplate reader (Tecan, Crailsheim, Germany) at405 nm. Results are shown in FIGS. 2-17.

TABLE 5 Composition and physico-chemical parameters of the benchmarkformulation RLX-165 and the LNP RLK-044 containing lipid KL22 of thepresent invention. The benchmark formulation was prepared according to apublished protocol (Nature Biotechnology 2010, 28: 172) with theexception of a slightly different PEG-lipid. Instead of PEG2000-c-DMA(methoxypolyethyleneglycol-carbamoyl-dimyristyloxy- propylamine),PEG2000-c-DMOG (α-[3′-(1,2-dimyristoyl-3-propanoxy)-carboxamide-propyl]-ω-methoxy-polyoxyethylene) was used. Amino-lipidHelper lipid Chol PEG2000-lipid Size LNP mol % mol % mol % mol % N/P(nm) Encap % RLX-165 57.1 XTC2 7.1 DPPC 34.4 1.4 DMOG 2.2 92 92 RLK-04450 KL22  10 DSPC 38.5 1.5 DMOG 7.6 92 80

TABLE 6 LNPs based on the amino-lipid KL10. Compositions,physico-chemical properties and serum FVII activity upon treatment with0.1 mg/kg siRNA directed against FVII. PEG2000- KL10 DSPC Chol c-DOMGSize Encap FVII No mol % mol % mol % mol % N/P (nm) (%) (%) 1 50 10 38.51.5 4.9 106 74 81 2 50 10 38.5 1.5 6.9 108 78 55 3 60 0 38.5 1.5 5.9 7936 54 4 50 10% C16 38.5 1.5 5.9 110 84 69 Diether PC 5 50 10 38.5 1.58.4 80 83 23 6 65 3.5 30 1.5 6.9 83 56 107 7 60 8.5 30 1.5 6.9 93 71 528 60 9.5 30 0.5 6.9 121 75 60 9 60 0 30 10 6.9 49 36 84 10 65 0 30 5 6.964 29 129 11 98.5 0 0 1.5 6.9 122 53 110 12 50 10% DMPC 38.5 1.5 6.9 9979 78 13 50 10% DPPC 38.5 1.5 6.9 100 81 43 14 50 10% DOPC 38.5 1.5 6.990 75 122 15 50 10% DLIPC 38.5 1.5 6.9 89 74 128 16 50 10% POPC 38.5 1.56.9 86 76 128 17 50 10% C18 38.5 1.5 6.9 96 86 46 Diether PC 18 50 10%C16 38.5 1.5 6.9 91 75 77 Lyso-PC 19 50 10% DOPE 38.5 1.5 6.9 88 69 12420 50 10% DOPG 38.5 1.5 6.9 92 39 77 21 50 10% SM 38.5 1.5 6.9 101 90 2722 50 10 38.5% 1.5 6.9 75 89 140 OChemsPC 23 50 10 38.5% DOPE 1.5 6.9 9989 153 24 50 10 38.5 1.5% mPEG- 6.9 129 88 97 1000 DM 25 50 10 38.5 1.5%mPEG- 6.9 120 88 108 2000 DS 26 50 10 38.5 1.5% PEG- 6.9 109 88 91 2000Chol 29 50 0 48.5% 1.5 6.9 111 86 81 4ME16:0PE XTC2 57.1 7.1% DPPC 34.41.4 2.2 95 92 65

TABLE 7 LNPs based on the amino-lipid KL22. Compositions,physico-chemical properties and serum FVII activity upon treatment with0.1 mg/kg siRNA directed against FVII. PEG2000-c- KL22 DSPC Chol DOMGSize No mol % mol % mol % mol % N/P (nm) Encap % FVII % 1 50 10 38.5 1.55.5 78 46 68 2 50 10 38.5 1.5 10 76 63 45 3 50 10 38.5 1.5 15 81 69 61 450 10 39.5 0.5 7.6 126 84 64 5 50 10 40 0 7.6 736 40 78 6 50 20 30 0 7.6414 9 nd 7 60 0 38.5 1.5 7.6 84 28 nd 8 60 8.5 30 1.5 7.6 92 36 80 9 4020 38.5 1.5 7.6 93 82 79 10 65 0 30 5 7.6 59 5 nd 11 98.5 0 0 1.5 7.6 950 nd 12 50 10% DMPC 38.5 1.5 7.6 78 57 92 13 50 10% DPPC 38.5 1.5 7.6 9047 73 14 50 10% DOPC 38.5 1.5 7.6 71 62 57 15 50 10% DLiPC 38.5 1.5 7.684 59 63 16 50 10% POPC 38.5 1.5 7.6 74 55 81 17 50 10% C18 38.5 1.5 7.696 61 103 Diether PC 18 50 10% C16 38.5 1.5 7.6 75 38 75 Lyso-PC 19 5010% DOPE 38.5 1.5 7.6 75 49 59 20 50 10% DOPG 38.5 1.5 7.6 80 48 131 2150 10% SM 38.5 1.5 7.6 92 64 68 22 50 10 38.5% 1.5 7.6 76 84 107OChemsPC 23 50 10 38.5% DOPE 1.5 7.6 93 75 127 24 50 10 38.5% DSPC 1.57.6 246 1 nd 25 50 10 38.5 1.5% mPEG- 7.6 122 86 112 1000 DM 26 50 1038.5 1.5% mPEG- 7.6 91 36 nd 5000 DM 27 50 10 38.5 1.5% mPEG- 7.6 119 86106 1000 DS 28 50 10 38.5 1.5% mPEG- 7.6 106 68 107 2000 DS 29 50 1038.5 1.5% PEG-1000 7.6 147 90 110 Chol 30 50 10 38.5 1.5% PEG-2000 7.6101 64 134 Chol XTC2 57.1 7.1% OPPC 34.4 1.4 2.2 95 92 44

TABLE 8 LNPs based on the amino-lipid KL25. Compositions,physico-chemical properties and FVII mRNA levels upon treatment with 0.1mg/kg siRNA directed against FVII. KL25 DSPC Cholesterol PEG2000c-DOMGSize No mol % mol % mol % mol % N/P (nm) Encap % FVII % Std 50 10 38.51.5 6.7 142 90 81 1 50 10 38.5% DOPE 1.5 6.7 nd — nd 2 50 10 40   0  6.7 nd — nd 3 50 10 38.5 1.5 5 109 31 nd 4 50 10 38.5 1.5 10 128 68 35 550 10 38.5 1.5 15 120 72 34 6 40 20 38.5 1.5 6.7 111 47 39 7 60  0 38.51.5 6.7 113 34 40 8 50 10% C16 38.5 1.5 6.7 132 65 67 Diether PC 9 5010% SM 38.5 1.5 6.7 123 58 51 10 50 10 38.5 1.5% mPEG-1000 6.7 177 59 47DM 11 50 10 38.5 1.5% mPEG-1000 6.7 122 80 50 DS 12 50 10 38.5 1.5%mPEG-2000 6.7 123 49 74 DS 13 50 10 38.5 1.5% PEG-1000 6.7 207  9 ndChol 14 50 10 38.5 1.5% PEG-2000 6.7 141 46 68 Chol

TABLE 9 LNPs based on the amino-lipid KL25. Compositions andphysico-chemical properties. PEG2000- KL25 DSPC Chol c-DOMG Size EncapNo mol % mol % mol % mol % N/P (nm) % std 50 10 38.5 1.5 6.7 142 90 1 5010 38.5% 1.5 6.7 nd — DOPE 2 50 10 40   0   6.7 nd — 3 50 10 38.5 1.5 5109 31 4 50 10 38.5 1.5 10 128 68 5 50 10 38.5 1.5 15 120 72 6 40 2038.5 1.5 6.7 111 47 7 60  0 38.5 1.5 6.7 113 34 8 50 10% C16 38.5 1.56.7 132 65 Diether PC 9 50 10% SM 38.5 1.5 6.7 123 58 10 50 10 38.5 1.5%mPEG- 6.7 177 59 1000 DM 11 50 10 38.5 1.5% mPEG- 6.7 122 80 1000 DS 1250 10 38.5 1.5% mPEG- 6.7 123 49 2000 DS 13 50 10 38.5 1.5% 6.7 207  9PEG-1000 Chol 14 50 10 38.5 1.5% 6.7 141 46 PEG-2000 Chol

TABLE 10 Tek mRNA levels in various organs upon treatment with LNPsbased on amino-lipid KL25. Kidney Lung Jejunum Spleen Muscle No % Tek %Tek % Tek % Tek % Tek std 41 43 72 47 77 3 20 21 25 31 34 4 18 17 27 2538 5 35 35 58 32 40 6 22 18 41 28 25 7 38 33 57 31 48 8 42 43 47 40 65 951 48 39 32 43 10 40 42 26 27 47 11 45 37 67 60 88 12 54 49 60 62 56 1354 64 47 48 73 14 53 52 54 51 68 saline 100 100 100 100 100 LNPscontained a pool of 5 siRNA of which one was directed against Tek (0.2mg/kg of each siRNA, total siRNA dose 1 mg/kg). Values are given as themean of two treated animals relative to saline treated animals.

TABLE 11 mRNA levels in liver after treatment with LNPs based onamino-lipid KL25. No % FVII % Clec4f % Rein % Tek % GFP std 81 51 32 5180 3 35 29 28 57 61 4 34 29 23 60 58 5 39 31 51 91 75 6 40 27 58 66 72 767 44 41 64 80 8 51 36 40 59 84 9 47 36 57 58 74 10 50 33 30 61 80 11 7446 43 65 82 12 68 59 55 66 89 13 65 63 55 59 72 14 55 42 17 67 70 saline100 100 100 100 100 LNPs contained a pool of 5 siRNAs directed againstthe targets listed in the table (0.2 mg/kg of each siRNA, total siRNAdose 1 mg/kg). Values are given as the mean of two treated animalsrelative to saline treated animals.

TABLE 12 Composition and physico-chemical properties of selected LNPsbased on amino-lipid KL10. KL10 PEG2000- mol DSPC Chol c-DOMG Size EncapNo % mol % mol % mol % N/P (nm) % 21 50 10% SM 38.5 1.5 6.9 92 64 23 5010 38.5 DOPE 1.5 6.9 93 75 29 50  0 48.5% 1.5 6.9 111 86 4ME16:0PE

TABLE 13 mRNA levels of targets expressed in the liver upon treatmentwith LNPs based on amino-lipid KL10. LNPs contained a pool of 5 siRNAdirected against the targets in the table below (0.5 mg/kg of eachsiRNA). LNP Animal Rein GFP Tek Clec4f FVII KL 10-21 K1 13% 82% 25% 19%102% K2 15% 104% 30% 13% 106% KL 10-23 L1 17% 10% 46% 136% 28% L2 14% 8%35% 113% 22% KL 10-29 H1 12% 77% 33% 5% 88% H2 13% 65% 33% 12% 86%Saline S1 95% 101% 100% 97% 101% S2 105% 99% 100% 103% 99%

The invention claimed is:
 1. A linear amino-lipid having the structure:


2. A lipid nanoparticle comprising an amino-lipid of claim 1 and anadditional lipid selected from the group consisting of: cationic lipid,helper lipid, and PEG-lipid.
 3. The lipid nanoparticle of claim 2further comprising a hydrophobic small molecule.
 4. The lipidnanoparticle of claim 2 further comprising a biologically activecompound.
 5. The lipid nanoparticle of claim 4 wherein the biologicallyactive compound is selected from the group consisting of: smallmolecule, peptide, protein, carbohydrate, nucleic acid, and lipid. 6.The lipid nanoparticle of claim 2, wherein the cationic lipid is a lipidcomprising a quaternary amine with a nitrogen atom having four organicsubstituents.
 7. The lipid nanoparticle of claim 2, wherein the helperlipid is a neutral zwitterionic lipid.
 8. The lipid nanoparticle ofclaim 3, wherein the hydrophobic small molecule is selected from thegroup consisting of: a sterol and a hydrophobic vitamin.
 9. The lipidnanoparticle of claim 3, wherein the hydrophobic small molecule ischolesterol.
 10. The lipid nanoparticle of claim 5, wherein the peptideis a cell penetrating peptide.
 11. The lipid nanoparticle of claim 5,wherein the protein is selected from the group consisting ofnucleoprotein, mucoprotein, lipoprotein, synthetic polypeptide, a smallmolecule linked to a protein, and glycoprotein.
 12. The lipidnanoparticle of claim 5, wherein the nucleic acid is in the form of asingle stranded or partially double stranded oligomer or a polymercomposed of ribonucleotides.
 13. The lipid nanoparticle of claim 5,wherein the nucleic acid is selected from the group consisting of miRNA,antisense oligonucleotides, siRNA, immune-stimulatory oligonucleotides,aptamers, ribozymes, and plasmids encoding a specific gene or siRNA.